Dermaseptin-type and piscidin-type antimicrobial peptides

ABSTRACT

Antimicrobial agents, including antimicrobial peptides (AMPs) and uses thereof. Compositions and methods of using dermaseptin-type and piscidin-type antimicrobial peptides that demonstrate activity and improved therapeutic indices against microbial pathogens. The peptide compositions demonstrate the ability to not only maintain or improve antimicrobial activity against bacterial pathogens including Gram-negative microorganisms  Acinetobacter baumannii  and  Pseudomonas aeruginosa , but also significantly decrease hemolytic activity against human red blood cells. Specificity determinants within the AMPS change selectivity from broad spectrum antimicrobial activity to AMPS with gram-negative selectivity.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/114,037, filed Jul. 25, 2016, now U.S. Pat. No. 10,221,222, which isa U.S. national stage application under 35 U.S.C. 371 of PCT ApplicationNo. PCT/US2015/012913, having an international filing date of Jan. 26,2015, which designated the United States, which PCT application claimedthe benefit of U.S. Application Ser. No. 61/931,528, filed on Jan. 24,2014, both of which are incorporated herein by reference in theirentirety.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “2848-173-PUS-1_Sequence_Listing_ST25.txt”, having asize in bytes of 7000 bytes, and created on Jan. 7, 2019. Theinformation contained in this electronic file is hereby incorporated byreference in its entirety pursuant to 37 CFR § 1.52(e)(5).

FIELD OF THE INVENTION

This application relates to the field of antimicrobial agents, includingantimicrobial peptides (AMPs). The disclosure further relates toembodiments including compositions and methods comprisingdermaseptin-type and piscidin-type antimicrobial peptides.

BACKGROUND

Emergence of antimicrobial resistance is becoming a very large publichealth threat and has been recognized by, amongst others, the WorldHealth Organization, the U.S. Congress Office of Technology Assessmentand the United Kingdom House of Lords. The urgency to develop newclasses of antimicrobial agents particularly against Gram-negativepathogens Acinetobacter baumannii and Pseudomonas aeruginosa, wasdemonstrated by the dramatic increases in the incidence ofantibiotic-resistant species in a recent study in Mexico. In one study,550 clinical isolates of A. baumannii and 250 clinical isolates of P.aeruginosa were analyzed for the prevalence of multi-drug resistance,and 74% of A. baumannii and 34% of P. aeruginosa were multi-drugresistant.

Antimicrobial peptides (AMPs) are widely distributed in nature andrepresent a promising class of new antimicrobial agents. AMPs arerapidly bactericidal and generally have broad-spectrum activity. It isdifficult for bacteria to develop resistance to AMPs because their modeof action involves nonspecific interactions with the cytoplasmicmembrane. In addition, enantiomeric forms of AMPs with all-D-amino acidshave shown equal activities to their all-L-enantiomers, suggesting thatthe antimicrobial mechanism of such peptides does not involve astereoselective interaction with a chiral enzyme or lipid or proteinreceptor. In addition, all D-peptides are resistant to proteolyticenzyme degradation, which enhances their potential as therapeuticagents. However, it is widely believed that native AMPs lack specificityand might be too toxic (including due to the ability to lyse mammaliancells, normally expressed as hemolytic activity against human red bloodcells) to be used for systemic treatment.

The Dermaseptins are a family of linear peptides, initially isolated in1991 from the skin of various tree-dwelling, South American frogs of thePhyllomedusa species. These amphipathic α-helical cationic antimicrobialfamily of peptides are structurally and functionally related. Theyexhibit rapid cytolytic activity against a variety of microorganismsincluding viruses, bacteria, protozoa, yeast and filamentous fungi.Unlike other Dermaseptin members, Dermaseptin S4, a 28-residue AMP,lyses erythrocytes at micromolar concentrations. (The Dermaseptin S4 wasoriginally identified as a 28-mer peptide, but a deletion of a singleresidue in the sequence by the inventors produced an active 27-mer. Foraccuracy, the two peptides are referred to herein as “Dermaseptin S4,27-mer”, and “Dermaseptin S4, 28-mer.” The sequences of these peptidesare shown in Table 5 and additional data regarding the two forms isshown in FIG. 3.) The HC₅₀, the peptide concentration that causes 50%lysis of human red blood cells, was approximately 1.4 μM using a threehour incubation time at 37° C. Very rapid kinetics (within seconds) forthe lysis of human red blood cells can be observed under a microscope.This hemolytic activity of these peptides is a significant disadvantagefor the therapeutic use of these peptides.

The Piscidin family comprises the most common group of AMPs in teleostfish. Piscidin 1 was first isolated in 2001 from hybrid striped bass(Morone saxatilis male×Morone chrysops female), where it is produced inmast cells (immune cells of uncertain function present in allvertebrates), skin, gill and gastrointestinal tract. Piscidin 1 is a22-residue amphipathic α-helical AMP rich in histidines andphenylalanines. Piscidin 1 has the highest biological activity among thefamily with broad-spectrum activity against antibiotic-resistantbacteria, filamentous fungi, yeasts, and viruses. This peptide, however,is not selective for bacterial versus mammalian cells, and causedhemolysis of human red blood cells with a HC₅₀ of 11˜20 μM within onehour at 37° C. Thus, both AMPs from different natural sources suffer thedose or drug-limiting toxicity of hemolytic activity.

Thus, a new class of antimicrobial agents with lower rates of resistanceand different microbial targets is urgently needed because of therapidly increasing resistance to classical antibiotics. Amphipathiccationic α-helical antimicrobial peptides (AMPs) represent such a classof compounds, but the toxicity of these compounds to mammalian cellsmust be overcome.

SUMMARY

The invention provides antimicrobial agents comprising peptides andpeptide-containing compositions. In related embodiments, the inventionprovides methods of inhibiting microorganisms. In certain embodiments,the invention provides methods of treating a subject by administering anantimicrobial peptide (AMP) or composition containing one or more AMPsdescribed herein. In embodiments, compositions and methods of AMPs aredescribed which demonstrate activity and improved therapeutic indicesagainst bacterial pathogens. In embodiments, peptide compositionsdemonstrate the ability to not only maintain or improve antimicrobialactivity against bacterial pathogens including Gram-negativemicroorganisms Acinetobacter baumannii and Pseudomonas aeruginosa, butalso significantly decrease the hemolytic activity against human redblood cells. Thus, improved therapeutic indices are achieved by AMPsdisclosed herein.

To overcome the significant mammalian toxicity of most of the knownAMPs, the inventors developed the design concept of the “specificitydeterminant” which, in embodiments described herein, refers tosubstituting positively charged residue(s) in the non-polar face ofamphipathic α-helical or cyclic β-sheet within antimicrobial peptides tocreate selectivity between eukaryotic and prokaryotic membranes; thatis, antimicrobial activity is maintained and hemolytic activity or celltoxicity to mammalian cells is decreased or eliminated.

The inventors selected Piscidin 1 and Dermaseptin S4 as examples ofnative AMPs to substitute one or two amino acid(s) to lysine(s) atdifferent positions in their non-polar faces to investigate and developthe application of the “specificity determinant” design concept toenhance or maintain antimicrobial activity and significantly improve thetherapeutic index.

This disclosure provides antimicrobial agents comprising peptidecompositions, as well as methods of inhibiting microorganisms. In anembodiment, this disclosure provides a method of treating a subject byadministering a composition as described herein. In embodiments,antimicrobial peptides (AMPs) are described which demonstrate activityand improved therapeutic indices against bacterial pathogens. Thepeptides may demonstrate the ability to not only maintain or improveantimicrobial activity against bacterial pathogens, includingGram-negative microorganisms Acinetobacter baumannii and Pseudomonasaeruginosa, but also significantly decrease the hemolytic activityagainst human red blood cells. Thus, improved therapeutic indices areachieved by the disclosed AMPs.

In an aspect, this disclosure provides an isolated peptide comprisingthe amino acid sequence of D-Dermaseptin S4 L7K/A14K(ALWMTLKKKVLKAKAKALNAVLVGANA, SEQ ID NO:9) or a variant or derivativethereof, and wherein the peptide does not have the sequence ofD-Dermaseptin S4 (27-mer) (ALWMTLLKKVLKAAAKALNAVLVGANA; SEQ ID NO:6).

In related aspects, this disclosure provides a variant of theD-Dermaseptin S4 L7K/A14K peptide, wherein the variant has one, two, orthree amino acid modifications shown in the peptides of Tables 1 and 5.

In another aspect, this disclosure provides a peptide comprising thesequences of D-Dermaseptin S4 L7K/A14K. In another aspect, thisdisclosure provides a peptide consisting of the sequences ofD-Dermaseptin S4 L7K/A14K.

In these embodiments, the peptides may have one or more improvedbiological properties relative to D-Dermaseptin S4 (27-mer), whereinsaid one or more properties are selected from the group consisting ofantimicrobial activity, hemolytic activity, stability, and therapeuticindex for a microorganism.

Another aspect of this disclosure provides a pharmaceutical compositioncomprising at least one of the antimicrobial peptides of thisdisclosure. In a specific embodiment, the pharmaceutical compositioncomprises a peptide consisting of the sequence of D-Dermaseptin S4L7K/A14K (SEQ ID NO:9).

Another embodiment provides a method of preventing or treating aninfection in a subject, wherein the method comprises the step ofadministering a therapeutically effective amount of a composition to thesubject, wherein the composition comprises at least one antimicrobialpeptide of embodiment 1 and a pharmaceutically acceptable carrier. Incertain embodiments, the microorganism is selected from the groupconsisting of gram-positive bacteria and gram-negative bacteria.

Another embodiment provides a method of inhibiting a microorganism, themethod comprising contacting the microorganism with a compositioncomprising at least one antimicrobial peptide of this disclosure.

Another embodiment provides an isolated peptide comprising the aminoacid sequence of at least the first 16 residues of D-Dermaseptin S4L7K/A14K (SEQ ID NO:9) or a variant or derivative thereof, and whereinthe peptide does not have the sequence of D-Dermaseptin S4 (SEQ IDNO:6).

Another embodiment provides an isolated peptide comprising the aminoacid sequence of the peptide selected from the group consisting of anyof the antimicrobial peptides of this disclosure, including a peptide ofTables 1 or 5, and a derivative or variant thereof, that maintainssubstantially similar antimicrobial activity compared to theD-Dermaseptin S4 L7K/A14K peptide.

One aspect of this disclosure provides an antimicrobial peptide (AMP)comprising an amino acid sequence having at least 85%, or at least 90%or at least 95% homology with a peptide selected from the groupconsisting of:

(SEQ ID NO: 2) NH₂-FFHHIFRPIVHVGKTIHRLVTG-amide; (SEQ ID NO: 3)NH₂-FFHHIFRGKVHVGKTIHRLVTG-amide (SEQ ID NO: 4)NH₂-FFHHIFRGIVHKGKTIHRLVTG-amide (SEQ ID NO: 5)NH₂-FFHHIFRGIVHVKKTIHRLVTG-amide (SEQ ID NO: 7)NH₂-ALWMTLKKKVLKAAAKALNAVLVGANA-amide (SEQ ID NO: 8)NH₂-ALWMTLLKKVLKAKAKALNAVLVGANA-amide (SEQ ID NO: 9)NH₂-ALWMTLKKKVLKAKAKALNAVLVGANA-amide (SEQ ID NO: 12)NH₂-ALWMTLKKKVLKAKAKAALNVALVGANA-amide (SEQ ID NO: 13)NH₂-ALWMTLKKKVLKAKAK-amide (SEQ ID NO: 14)NH₂-ALWMTKLKKVLKAKAKALNAVLVGANA-amide (SEQ ID NO: 15)NH₂-ALWMTLKKKVLKAKAKALNAVLSGANA-amide (SEQ ID NO: 16)NH₂-ALWMTLKKKVLKAKAKALNAVLKGANA-amide (SEQ ID NO: 17)NH₂-ALWMTLKKKVLKAKAKALNAVLAGVNA-amide (SEQ ID NO: 18)NH₂-ALWMTLKKKVLKAKAKLLNAVLVGANA-amide (SEQ ID NO 19)NH₂-ALWMTLKKKVLKAKAKALNAVLVGANA-amide, and (SEQ ID NO: 20)NH₂-ALWMTLKKKVLKAKAKLLNAVLVGLNA-amide,or functional analogues, derivatives or fragments thereof, or apharmaceutically-acceptable salt thereof.

In certain embodiments, the AMP comprises the sequence of any one of SEQID NOs.: 2-5, 7-9, and 12-20. In related embodiments, the AMP consistsof the sequence of any one of SEQ ID NOs.: 2-5, 7-9, and 12-20. Inspecific embodiments, the AMP comprises the sequence of any one of SEQID NOs.:2-5. In specific embodiments, the AMP comprises the sequence ofany one of SEQ ID NOs.:7-9. In specific embodiments, the AMP comprisesthe sequence of any one of SEQ ID NOs.:12-20. In a preferred embodiment,the amino acid sequence of the AMP comprises the sequence of SEQ IDNO:9. In a related embodiment, the amino acid sequence of the AMPconsists of the sequence of SEQ ID NO:9. In these embodiments, the AMPinhibits propagation of a prokaryote. In certain embodiments, theprokaryote is a Gram negative bacterium, which may include at least oneof A. baumannii and P. aeruginosa. In other embodiments, the prokaryoteis a Gram-positive bacterium which may include methicillin-resistantStaphylococcus aureus (MRSA).

In certain embodiments, the AMP exhibits at least a 3-fold increasedselectivity for Gram negative bacteria over Gram-positive bacteriacompared to the selectivity of SEQ ID NO:1.

In related embodiments, the AMP exhibits at least a 50-fold increasedselectivity for Gram negative bacteria over Gram-positive bacteriacompared to the selectivity of SEQ ID NO. 1.

In certain embodiments, the AMP exhibits at least a 3-fold increasedselectivity for Gram negative bacteria over Gram-positive bacteriacompared to the selectivity of SEQ ID NO:6.

In related embodiments, the AMP exhibits at least a 100-fold increasedselectivity for Gram negative bacteria over Gram-positive bacteriacompared to the selectivity of SEQ ID NO:6.

In certain embodiments the AMP exhibits at least a 3-fold increasedselectivity for prokaryotic cells over eukaryotic cells compared to theselectivity of SEQ ID NO: 1. In related embodiments, the AMP exhibits atleast a 30-fold increased selectivity for prokaryotic cells overeukaryotic cells compared to the selectivity of SEQ ID NO:1. In certainembodiments, the AMP exhibits at least a 10-fold increased selectivityfor prokaryotic cells over eukaryotic cells compared to the selectivityof SEQ ID NO:6. In related embodiments, the AMP exhibits at least a400-fold increased selectivity for prokaryotic cells over eukaryoticcells compared to the selectivity of SEQ ID NO:6. In relatedembodiments, the AMP exhibits at least a 700-fold increased selectivityfor prokaryotic cells over eukaryotic cells compared to the selectivityof SEQ ID NO:6.

In certain embodiments, the AMP exhibits at least a 15-fold decreasedhemolysis of human red blood cells compared to hemolysis exhibited bySEQ ID NO: 1. In related embodiments, the AMP exhibits at least a50-fold decreased hemolysis of human red blood cells compared tohemolysis exhibited by SEQ ID NO:1. In certain embodiments, the AMPexhibits at least a 10-fold decreased hemolysis of human red blood cellscompared to hemolysis exhibited by SEQ ID NO:6. In related embodiments,the AMP exhibits at least a 400-fold decreased hemolysis of human redblood cells compared to hemolysis exhibited by SEQ ID NO:6.

In these embodiments, the antibiotic resistant prokaryote may be agram-negative resistant Acinetobacter baumannii or Pseudomonasaeruginosa pathogen and an antibiotic sensitive prokaryote may be anAcinetobacter baumannii or Pseudomonas aeruginosa sensitive pathogen.Alternatively or additionally, the antibiotic resistant prokaryote maybe a colistin (polymyxin E) resistant gram-negative pathogen or acolistin sensitive gram-negative pathogen.

Another aspect of this disclosure provides a pharmaceutical compositioncomprising at least one AMP of this disclosure and a pharmaceuticallyacceptable carrier. In one embodiment, the pharmaceutical composition isa mono-phasic pharmaceutical composition suitable for parenteral or oraladministration consisting essentially of a therapeutically-effectiveamount of at least one AMP of this disclosure, and a pharmaceuticallyacceptable carrier.

Another aspect of this disclosure provides methods of preventing ortreating a microbial infection comprising administering to a subject inneed thereof a therapeutically effective amount of at least one AMP ofthis disclosure, or a pharmaceutical composition comprising the same.

In these methods, the microbial infection may be the result of aninfecting bacteria, fungi, virus, or protozoa. In certain embodiments,the microbial infection is a bacterial infection. In specificembodiments, the bacterial infection is a Gram negative bacterialinfection. In related embodiments, the bacterial infection is anantibiotic resistant bacterial infection. In certain embodiments, theinfecting microorganism is at least one of Escherichia coli, Pseudomonasaeruginosa, Salmonella spp., Hemophilus influenza, Neisseria spp.,Vibrio cholerae, Vibrio parahaemolyticus and Helicobacter pylori. Inspecific embodiments, the infecting microorganism is the yeast Candidaalbicans. In certain embodiments, the infecting microorganism is atleast one of measles virus, herpes simplex virus (HSV-1 and -2), herpesfamily members (HIV, hepatitis C, vesicular, stomatitis virus (VSV),visna virus, and cytomegalovirus (CMV). In another embodiment, theinfecting microorganism is at least one of Giardia, Acinetobacterbaumannii and Pseudomonas aeruginosa. In a specific embodiment, theinfecting microorganism is multi-drug resistant Pseudomonas aeruginosaor Acinetobacter baumannii bacteria.

In these methods, the administration of the peptide or pharmaceuticalcomposition may be made by an administration route selected from oral,topical, intravenous, intraperitoneal, intramuscular, intradermal,intrasternal, intraarticular injection, or infusion. In certainembodiments, the peptide or pharmaceutical composition is administeredin conjunction with one or more additional antimicrobial agents.

Another embodiment is a method of preventing a microbial infection in anindividual at risk of developing an infection comprising administeringan effective amount of at least one AMP of this disclosure or apharmaceutical composition comprising the same to an individual in needthereof. In certain embodiments, the individual is a surgical patient.In related embodiments, the individual is a hospitalized patient.

A method of combating a bacterial infection in a patient, comprisingapplying at least one AMP of this disclosure or a pharmaceuticalcomposition comprising the same to a body surface of the patient. In aspecific embodiment, the body surface is a wound. In a specificembodiment, the composition is applied following an operation orsurgery.

Another embodiment provides at least one AMP of this disclosure or apharmaceutical composition comprising the same for use in the treatmentof a microbial infection. A related embodiment provides the use of atleast one peptide of this disclosure or a pharmaceutical compositioncomprising the same in the manufacture of a medicament for theprevention or treatment of a microbial infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows helical wheel (upper panel) and helical net (lower panel)representations of D-Piscidin 1 and related peptide analogs listed inTable 1. The one-letter code is used for amino acid residues. ‘D’denotes that all residues in the peptides are in the D-conformation.Positively charged residues (Lys and Arg) are darkly shaded on the leftside of the helical wheel and the right side of the helical net. Largehydrophobes (Val, lie, Leu and Phe) are lightly shaded on the right sideof the helical wheel and the left side of the helical net. The“specificity determinant” amino acid change is denoted by shadedtriangles in the three D-Piscidin 1 analogs of the invention. In thehelical wheel, the non-polar face is indicated as an open arc (doublelines) and the polar face is shown as a solid arc (single thick line).In the helical net, the residues on the polar face are boxed and theresidues on the non-polar face are circled. The i→i+3 and i→i+4potential hydrophobic interactions along the helix are shown as blackbars. The i→i+3 and i→i+4 potential electrostatic repulsions betweenpositively charged residues along the helix are shown as dotted bars.

FIG. 2 shows helical wheel (upper panel) and helical net (lower panel)representation of D-Dermaseptin S4 and analogs, which are listed inTable 1. The one-letter code is used for amino acid residues. ‘D’denotes that all residues in the peptides are in the D conformation.Positively charged residues (Lys and Arg) are darkly shaded on the rightside of the helical wheel and the upper portion of the helical net.Large hydrophobes (Val, lie, Leu and Phe) are lightly shaded on theupper and left side of the helical wheel and both the upper and lowerportions of the helical net. The “specificity determinant” amino acidchange is denoted by shaded triangles in the three D-Dermaseptin S4analogs of the invention. In the helical wheel, the non-polar face isindicated as an open arc and the polar face is shown as a solid arc. Inthe helical net, the residues on the polar face are boxed and theresidues on the non-polar face are circled. The i→i+3 and i→i+4potential hydrophobic interactions along the helix are shown as blackbars. The i→i+3 and i→i+4 potential electrostatic repulsions betweenpositively charged residues along the helix are shown as dotted bars.

FIG. 3 shows Helical wheel (upper panel) and helical net (lower panel)representation of D-Dermaseptin S4 (27 mer) and D-Dermaseptin S4 (28mer). The one-letter code is used for amino acid residues. ‘D’ denotesthat all residues in the peptides are in the D conformation. Positivelycharged residues (Lys) are darkly shaded, while large hydrophobes (Val,Leu, Met and Trp) are lightly shaded. In the helical wheel, thenon-polar face is indicated as an open arc and the polar face is shownas a solid arc. In the helical net, the residues on the polar face areboxed and the residues on the non-polar face are circled. The i→i+3 andi→i+4 potential hydrophobic interactions along the helix are shown asblack bars. The i→i+3 and i→i+4 potential electrostatic repulsionsbetween positively charged residues along the helix are shown as dottedbars.

FIGS. 4A-4C show CD spectra of D-Pisicidin 1 and D-Dermaseptin S4analogs of the invention. FIG. 4A shows the CD spectra of D-Pisicidin 1analogs in aqueous benign buffer (100 mM KCl, 50 mM NaH₂PO₄/Na₂HPO₄ atpH 7.0), 5° C. (closed symbols) and in the presence ofbuffer-trifluoroethand (TFE) (1:1, v/v) (open symbols). FIG. 4B showsthe CD spectra of D-Dermaseptin S4 analogs in aqueous benign buffer (100mM KCl, 50 mM NaH₂PO₄/Na₂HPO₄ at pH 7.0), 5° C., and FIG. 4C showsD-Dermaseptin S4 analogs in the presence of buffer-trifluoroethanol(TFE) (1:1, v/v).

FIG. 5 shows a proposed mechanism for temperature profiling by RP-HPLC.Only the folded monomeric α-helix is bound to the hydrophobicreversed-phase matrix. During partitioning at low temperature, there isan equilibrium between monomer and dimer in the mobile phase. At somehigher temperature during partitioning there is only monomer present inthe mobile phase. This method measures the self-association parameterfor any amphipathic molecule, as demonstrated for D-Dermaseptin S4 andits analogs in FIG. 6.

FIG. 6 shows D-Dermaseptin S4 analogs self-association ability asmonitored by temperature profiling in RP-HPLC. In panel A of FIG. 6, theretention times of peptides are normalized to 5° C. through theexpression (t_(R) ¹−t_(R) ⁵), where t_(R) ¹ is the retention time at aspecific temperature of an antimicrobial peptide or control peptide C,and t_(R) ⁵ is the retention time at 5° C. In panel B of FIG. 6, theretention behavior of the peptides was normalized to that of controlpeptide C through the expression (t_(R)−t_(R) ⁵ for peptides)−(t_(R)¹−t_(R) ⁵ for control peptide C). The maximum change in retention timefrom the control peptide C defines the peptide association parameter,denoted PA (Table 2). The sequences of the peptides and the random coilcontrol peptide (C) are shown in Table 1.

FIG. 7 shows the hemolytic activity of peptide D-Piscidin 1 and analogs(panel A of FIG. 7) and D-Dermaseptin S4 and analogs (panel B of FIG. 7)after 18 hours of incubation time at 37° C. The concentration-responsecurves of peptides for percentage lysis of human red blood cells (hRBC)are shown. The control for 100% hemolysis was a sample of erythrocytestreated with water. The peptide concentration is reported as micromolar(μM).

FIG. 8 shows the comparison of hemolytic activity of peptide D-Piscidin1 G8P and D-Piscidin 119K after 1 hour or 18 hours treatment. Theconcentration-response curves of peptides for percentage lysis of humanred blood cells (hRBC) are shown. The control for 100% hemolysis was asample of erythrocytes treated with 0.1% Triton-X 100. The peptideconcentration is in μM.

FIGS. 9A-9B illustrate structural configurations of the peptideD-Dermaseptin S4 L7K, A14K. FIG. 9A shows a helical wheel configurationwith non-polar and polar faces indicated as an open arc (double lines)and solid arc (single thick line), respectively. FIG. 9B shows thehelical net representations of the polar and non-polar faces of thepeptide. The one-letter code is used for amino acid residues. ‘D’denotes that all residues in the peptides are in the D conformation.Positively charged residues (Lys) are shaded black, large hydrophobes(Val, Leu, Met and Trp) have hatched shading. The “specificitydeterminants” are denoted by black triangle(s). In the helical netrepresentations of FIG. 9B, the residues on the polar face are boxed andthe residues on the non-polar face are circled. The i→i+3 and i→i+4potential hydrophobic interactions along the helix are shown as solidblack bars. The i→i+3 and i→i+4 potential electrostatic repulsionsbetween positively charged residues along the helix are shown as dottedbars.

DETAILED DESCRIPTION

In general the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art.

As used herein, the singular forms “a”, “an”, and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andequivalents thereof known to those skilled in the art, and so forth. Aswell, the terms “a” (or “an”), “one or more” and “at least one” can beused interchangeably herein. It is also to be noted that the terms“comprising”, “including”, and “containing” can be used interchangeably.The expression “of any of claims XX-YY” (wherein XX and YY refer toclaim numbers) is intended to provide a multiple dependent claim in thealternative form, and in some embodiments is interchangeable with theexpression “as in any one of claims XX-YY.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the pertinent art.

Whenever a range of values is given in the specification, for example, atemperature range, a time range, or a composition or concentrationrange, all intermediate ranges and subranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure. As used herein, ranges specifically include the valuesprovided as endpoint values of the range. For example, a range of 1 to100 specifically includes the end point values of 1 and 100. It will beunderstood that any subranges or individual values in a range orsubrange that are included in the description herein can be excludedfrom the claims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be optionally replaced with either of the other twoterms, thus describing alternative aspects of the scope of the subjectmatter. The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

The following definitions are provided to clarify use of these terms inthe context of embodiments of the invention.

When used herein, the term “amino acid” is intended to refer to anynatural or unnatural amino acid, whether made naturally orsynthetically, including any such in L- or D-configuration. The term canalso encompass amino acid analog compounds used in peptidomimetics or inpeptoids. The term can include a modified or unusual amino acid or asynthetic derivative of an amino acid, e.g. diamino butyric acid anddiamino propionic acid and the like. In embodiments, of antimicrobialpeptides comprise amino acids linked together by peptide bonds. Thepeptides are in general in alpha helical conformation under hydrophobicconditions. Sequences are conventionally given from the amino terminusto the carboxyl terminus. Unless otherwise noted, the amino acids areL-amino acids. When all the amino acids are of L-configuration, thepeptide is said to be an L-enantiomer. When all the amino acids are ofD-configuration, the peptide is said to be a D-enantiomer.

The term “hemolytic concentration—50 or HC₅₀” refers to the peptideconcentration that causes 50% hemolysis of erythrocytes after 18 h. HC₅₀was determined from a plot of percent lysis versus peptide concentration(μM). For comparison, the inventors also determined the hemolyticactivity after 1 hour at 37° C. Hemolysis can be determined with redblood cells (RBC) from various species including human red blood cells(hRBC).

The term “therapeutic index” (TI) is the ratio of HC₅₀ over minimalinhibitory concentration (MIC) of an antimicrobial agent. Larger valuesgenerally indicate greater antimicrobial specificity.

The term “stability” can refer to an ability to resist degradation, topersist in a given environment, and/or to maintain a particularstructure. For example, a peptide property of stability can indicateresistance to proteolytic degradation and to maintain an alpha-helicalstructural conformation.

The following abbreviations are used herein: A, Ala, Alanine; M, Met,Methionine; C, Cys, Cysteine, D, Asp, Aspartic Acid, E, Glu, GlutamicAcid; F, Phe, Phenylalanine; G, Gly, Glycine; H, His, Histidine; I, lie,Isoleucine; K, Lys, Lysine; L, Leu, Leucine; N, Asn, Asparagine, P, Pro,Proline; Q, Glu, Glutamine; R, Arg, Arginine; S, Ser, Serine; T, Thr,Threonine; V, Val, Valine; W, Trp, Tryptophan; Y, Tyr, Tyrosine;RP-HPLC, reversed-phase high performance liquid chromatography, MIC,minimal inhibitory concentration; HC₅₀ hemolytic concentration—50; CD,circular dichroism spectroscopy; TFE, 2,2,2-trifluoroethanol; TFA,trifluoroacetic acid; RBC, red blood cells; hRBC, human red blood cells.

The term “antimicrobial activity” refers to the ability of a peptideembodiment to modify a function or metabolic process of a targetmicroorganism, for example so as to at least partially affectreplication, vegetative growth, toxin production, survival, viability ina quiescent state, or other attribute. In an embodiment, the termrelates to inhibition of growth of a microorganism. In a particularembodiment, antimicrobial activity relates to the ability of a peptideto kill at least one bacterial species. In a particular embodiment, thebacterial species is selected from the group consisting of gram-positiveand gram-negative bacteria. In an embodiment, the term can be manifestedas microbicidal or microbistatic inhibition of microbial growth.

The phrase “improved biological property” is meant to indicate that atest peptide exhibits less hemolytic activity and/or betterantimicrobial activity, or better antimicrobial activity and/or lesshemolytic activity, compared to the control peptide (e.g., D-Piscidin 1or D-Dermaseptin S4), when tested by the protocols described herein orby any other art-known standard protocols. In general, the improvedbiological property of the peptide is reflected in the therapeutic index(TI) value which is better than that of the control peptide.

The term “microorganism” herein refers broadly to bacteria, fungi,viruses, and protozoa. In particular, the term is applicable for amicroorganism having a cellular or structural component of a lipidbilayer membrane. In specific embodiments, the membrane is a cytoplasmicmembrane. Pathogenic bacteria, fungi, viruses, and protozoa as known inthe art are generally encompassed. Bacteria can include gram-negativeand gram-positive bacteria in addition to organisms classified in ordersof the class Mollicutes and the like, such as species of the Mycoplasmaand Acholeplasma genera. Specific examples of potentially sensitivegram-negative bacteria include, but are not limited to, Escherichiacoli, Pseudomonas aeruginosa. Salmonella spp., Hemophilus influenza,Neisseria spp., Vibrio cholerae, Vibrio parahaemolyticus andHelicobacter pylori. Examples of potentially sensitive gram-positivebacteria include, but are not limited to, Staphylococcus aureus,Staphylococcus epidermidis, Streptococcus agalactiae, Group AStreptococcus, Streptococcus pyogenes, Enterococcus faecalis, Group BGram-positive Streptococcus, Corynebacterium xerosis, and Listeriamonocytogenes. Examples of potentially sensitive fungi include yeastssuch as Candida albicans. Examples of potentially sensitive virusesinclude measles virus, herpes simplex virus (HSV-1 and -2), herpesfamily members (HIV, hepatitis C, vesicular, stomatitis virus (VSV),visna virus, and cytomegalovirus (CMV). Examples of potentiallysensitive protozoa include Giardia.

“Therapeutically effective amount” as used herein, refers to an amountof formulation, composition, or reagent in a pharmaceutically acceptablecarrier or a physiologically acceptable salt of an active compound thatis of sufficient quantity to ameliorate the undesirable state of thepatient, animal, material, or object so treated. “Ameliorate” refers toa lessening of the detrimental effect of the disease state or disorder,or reduction in contamination, in the receiver of the treatment.

“Pharmaceutical agent or drug” as used herein, refers to a chemicalcompound or composition capable of inducing a desired therapeutic orprophylactic effect when properly administered to a subject.

“Pharmaceutically acceptable carrier” as used herein, refers toconventional pharmaceutical carriers useful in the methods disclosedherein. Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 15th Edition (1975), describes compositionsand formulations suitable for pharmaceutical delivery of TCR peptidesand additional pharmaceutical agents. In general, the nature of thecarrier will depend on the particular mode of administration beingemployed. For instance, parenteral formulations usually compriseinjectable fluids that include pharmaceutically and physiologicallyacceptable fluids such as water, physiological saline, balanced saltsolutions, aqueous dextrose, glycerol or the like as a vehicle. Forsolid compositions (e.g., powder, pill, tablet, or capsule forms),conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, salts,amino acids, and pH buffering agents and the like, for example sodium orpotassium chloride or phosphate, Tween, sodium acetate or sorbitanmonolaurate.

Antimicrobial peptides (AMPs) of the invention have antimicrobialactivity by themselves or when covalently conjugated or otherwisecoupled or associated with another molecule, e.g., polyethylene glycolor a carrier protein such as bovine serum albumin, so long as thepeptides are positioned such that they can come into contact with a cellor unit of the target microorganism. These peptides may be modified bymethods known in the art provided that the antimicrobial activity is notdestroyed or substantially compromised.

In embodiments of the invention, peptide compositions may be isolated orpurified. In embodiments, a peptide is synthetic and can be produced bypeptide synthesis techniques or by recombinant expression technology asunderstood in the art. As used herein, the term “purified” can beunderstood in embodiments to refer to a state of enrichment or selectiveenrichment of a particular component relative to an earlier state ofcrudeness or constituency of another component. In embodiments, the termcan be considered to correspond to a material that is at least partiallypurified as opposed to a state of absolute purity. For example in aparticular embodiment, a peptide composition can be considered purifiedeven if the composition does not reach a level of one hundred percentpurity with respect to other components in the composition.

As used herein, the term “specificity determinant(s)” refers to certainamino acid residues in an AMP of the invention. In particularembodiments, the term refers to positively charged residue(s) in thenon-polar face of AMPs that could decrease hemolytic activity/toxicitybut increase or maintain the same level of antimicrobial activity, thusincreasing the therapeutic index of the AMP.

Exemplary antimicrobial pedptides of the invention are listed inTable 1. Additional antimicrobial pedptides of the invention are listedin Table 5.

TABLE 1 Piscidin-type and Dermaseptin-type peptides Peptide Name^(a)Length Sequence^(b) SEQ ID NO. MW D-Piscidin 1 22NH₂-FFHHIFRGIVHVGKTIHRLVTG-amide  1 2571 D-Piscidin 1 G8P^(c) 22NH₂-FFHHIFRPIVHVGKTIHRLVTG-amide  2 2611 D-Piscidin 1 I9K 22NH₂-FFHHIFRGKVHVGKTIHRLVTG-amide  3 2586 D-Piscidin 1 V12K 22NH₂-FFHHIFRGIVHKGKTIHRLVTG-amide  4 2600 D-Piscidin 1 G13K 22NH₂-FFHHIFRGIVHVKKTIHRLVTG-amide  5 2642 D-Dermaseptin S4 27NH₂-ALWMTLLKKVLKAAAKALNAVLVGANA-amide  6 2778 D-Dermaseptin S4 L7K 27NH₂-ALWMTLKKKVLKAAAKALNAVLVGANA-amide  7 2794 D-Dermaseptin S4 A14K 27NH₂-ALWMTLLKKVLKAKAKALNAVLVGANA-amide  8 2837 D-Dermaseptin S4 L7K, A14K27 NH₂-ALWMTLKKKVLKAKAKALNAVLVGANA-amide  9 2851 Control C^(d) 18Ac-ELEKGGLEGEKGGKELEK-amide 10 — ^(a)The D- denotes that all amino acidresidues in each peptide are in the D conformation. ^(b)Peptidesequences are shown using the one-letter code for amino acid residues;Ac denotes N^(α)-acetyl and amide denotes C^(α)-amide. The “specificitydeterminant(s)”, Lys residues incorporated in the non-polar face arebolded. ^(c)The L-Piscidin 1 G8P was previously reported as a selectivepeptide. ^(d)This peptide is a random coil peptide in the allL-conformation used as a control for reversed-phase chromatographytemperature profiling to examine peptide self-association.Compositions of the Invention

When employed as pharmaceuticals, especially as antimicrobial agentsadministered to mammals, the AMPs of the invention are administered inthe form of pharmaceutical compositions. These compounds can beadministered by a variety of routes including oral, rectal, transdermal,subcutaneous, intravenous, intramuscular, and intranasal. Suchpharmaceutical compositions are prepared in a manner well known in thepharmaceutical art and comprise at least one AMP of the invention.

The pharmaceutical compositions of the present invention contain, as theactive ingredient, one or more of the AMPs of the invention, associatedwith pharmaceutically acceptable formulations. In making thecompositions of this invention, the active ingredient is usually mixedwith an excipient, diluted by an excipient or enclosed within a carrierwhich can be in the form of a capsule, sachet, paper or other container.An excipient is usually an inert substance that forms a vehicle for adrug. When the excipient serves as a diluent, it can be a solid,semi-solid, or liquid material, which acts as a vehicle, carrier ormedium for the active ingredient. Thus, the compositions can be in theform of solutions, syrups, aerosols (as a solid or in a liquid medium),ointments containing, for example, up to 30% by weight of the activecompound, soft and hard gelatin capsules, suppositories, sterileinjectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill active compoundsof the invention to provide the appropriate particle size prior tocombining with the other ingredients. If the antimicrobial peptide issubstantially insoluble, it ordinarily is milled to a particle size ofless than 200 mesh. If the compound(s) is substantially w ater soluble,the particle size is normally adjusted by milling to provide asubstantially uniform distribution in the formulation, e g. about 40mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, gum Arabic, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, andmethylcellulose. The formulations can additionally include: lubricatingagents such as talc, magnesium stearate, and mineral oil; wettingagents; emulsifying and suspending agents; preserving agents such asmethyl- and propylhydroxy-benzoates; sweetening agents; and flavoringagents. The compositions of the invention can be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to the patient by employing procedures known in theart.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, powders, granules or as asolution or a suspension in an aqueous or non-aqueous liquid, or anoil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), and the like, each containing a predeterminedamount of a compound or compounds of the present invention as an activeingredient. A compound or compounds of the present invention may also beadministered as bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonosterate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may be employed as fillers in soft andhard-filled gelatin capsules using such excipients as lactose or milksugars, as well as high molecular weight polyethylene glycols and thelike.

A tablet may be made by compression or molding optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxy propylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicroencapsulated form.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, w ater or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose,aluminum metahydroxide, bentonite, agar-agar and tragacanth, andmixtures thereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, poly ethylene glycol, asuppository wax or salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound. Formulations of thepresent invention which are suitable for vaginal administration alsoinclude pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate.

Dosage forms for the topical or transdermal administration of compoundsof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, drops and inhalants. The activeingredient may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any buffers, orpropellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive ingredient, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active ingredient,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and poly amide powder or mixtures of these substances.Sprays can additionally contain customary propellants such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of compounds of the invention to the body. Such dosage formscan be made by dissolving, dispersing or otherwise incorporating one ormore compounds of the invention in a proper medium, such as anelastomeric matrix material. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate of such fluxcan be controlled by either providing a rate-controlling membrane ordispersing the compound in a polymer matrix or gel.

Pharmaceutical formulations include those suitable for administration byinhalation or insufflation or for nasal or intraocular administration.For administration to the upper (nasal) or lower respiratory tract byinhalation, the compounds of the invention are conveniently deliveredfrom an insufflator, nebulizer or a pressurized pack or other convenientmeans of delivering an aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecomposition may take the form of a dry powder, for example, a powder mixof one or more compounds of the invention and a suitable powder base,such as lactose or starch. The powder composition may be presented inunit dosage form in, for example, capsules or cartridges, or, e.g.,gelatin or blister packs from which the powder may be administered withthe aid of an inhalator, insufflator or a metered-dose inhaler.

For intranasal administration, compounds of the invention may beadministered by means of nose drops or a liquid spray, such as by meansof a plastic bottle atomizer or metered-dose inhaler. Typical ofatomizers are the Mistometer (Wintrop) and Medihaler (Riker).

Drops, such as eye drops or nose drops, may be formulated with anaqueous or nonaqueous base also comprising one or more dispersingagents, solubilizing agents or suspending agents. Liquid spray s areconveniently delivered from pressurized packs. Drops can be delivered bymeans of a simple eye dropper-capped bottle or by means of a plasticbottle adapted to deliver liquid contents dropwise by means of aspecially shaped closure.

Pharmaceutical compositions of this invention suitable for parenteraladministrations comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, solutes which render the formulation isotonicwith the blood of the intended recipient or suspending or thickeningagents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monosterate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drug isaccomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

Suitable alkalinizing agents include alkali metal salts and alkalineearth metal salts. The alkali metal salts include sodium carbonate,sodium hydroxide, sodium silicate, disodium hydrogen orthophosphate,sodium aluminate, and other suitable alkali metal salts or mixturesthereof. Suitable alkaline metal salts include calcium carbonate,calcium hydroxide, magnesium carbonate, magnesium hydroxide, magnesiumsilicate, magnesium aluminate, aluminum magnesium hydroxide or mixturethereof. More particularly, calcium carbonate, potassium bicarbonate,calcium hydroxide, and/or sodium carbonate may be used as alkalinizingagents to obtain a formulation pH within the desired pH range of pH 8 topH 13. The concentration of the alkalinizing agent is selected to obtainthe desired pH, varying from about 0.1% to about 30%, by weight, andmore preferably from about 12.5% to about 30%, by weight, of the totalweight of the dosage formulation.

Suitable antioxidants may be selected from amongst one or morepharmaceutically acceptable antioxidants known in the art. Examples ofpharmaceutically acceptable antioxidants include butylatedhydroxyanisole (BHA), sodium ascorbate, butylated hydroxytoluene (BHT),sodium sulfite, citric acid, malic acid and ascorbic acid. Theantioxidants may be present in the dosage formulations of the presentinvention at a concentration between about 0.001% to about 5%, byweight, of the dosage formulation.

Suitable chelating agents may be selected from amongst one or morechelating agents known in the art. Examples of suitable chelating agentsinclude disodium edetate (EDTA), edetic acid, citric acid andcombinations thereof. The chelating agents may be present in aconcentration between about 0.001% and about 5%, by weight, of thedosage formulation.

Methods for Preventing and Treating Microbial Infection

Another aspect of the invention provides methods for preventing andtreating a microbial infection. These methods include administering to asubject in need thereof a therapeutically effective amount of a peptideor composition of the invention that kills or inhibits the growth ofinfectious microbes, thereby inhibiting or treating the microbialinfections.

In certain embodiments, the infectious microbes include at least one ofbacteria, fungi, viruses, and protozoa. In certain embodiments, theinfecting microorganism includes gram-negative and/or gram-positivebacteria in addition to organisms classified in orders of the classMollicutes and the like, such as species of the Mycoplasma andAcholeplasma genera. In specific embodiments, the infectingmicroorganism is a gram-negative bacteria, which may include, but is notlimited to, Escherichia coli, Pseudomonas aeruginosa. Salmonella spp.,Hemophilus influenza, Neisseria spp., Vibrio cholerae, Vibrioparahaemolyticus and Helicobacter pylori.

In specific embodiments, the infecting microorganism is a gram-positivebacteria, including, but are not limited to, Staphylococcus aureus,Staphylococcus epidermidis, Streptococcus agalactiae, Group AStreptococcus. Streptococcus pyogenes, Enterococcus faecalis. Group BGram-positive Streptococcus, Corynebacterium xerosis, and Listeriamonocytogenes.

In specific embodiments, the infecting microorganism is a fungi,including, but not limited to, yeasts such as Candida albicans.

In specific embodiments, the infecting microorganism is a virus,including, but not limited to, measles virus, herpes simplex virus(HSV-1 and -2), herpes family members (HIV, hepatitis C, vesicular,stomatitis virus (VSV), visna virus, and cytomegalovirus (CMV).

In specific embodiments, the infecting microorganism is a protozoa,including, but not limited to Giardia.

In these embodiments, the antimicrobial peptides administered,preferably as a component of a pharmaceutical composition, can include asingle peptide of Tables 1 or 5, or multiple peptides of Tables 1 and/or5. The peptides may include peptides having at least 85%, or at least90%, or at least 95% homology to a peptide sequence of those peptidesset forth in Tables 1 and 5, and which effectively treat or prevent amicrobial infection. The peptides may include fragments of the peptidesof Tables 1 and 5 that retain the ability to effectively treat orprevent a microbial infection.

The peptides may include peptides having at least one amino acidsubstitution of SEQ ID NOs: 3-5 and 7-9, and which retain theantimicrobial activity of D-Dermaseptin S4 and/or D-Piscidin 1. Anexemplary peptide includes the amino acid sequence set forth in SEQ IDNO:9. Appropriate peptides to use in the methods disclosed herein can bedetermined by those skilled in the art.

Therapeutical AMPs of this disclosure can be administered by a number ofroutes, including parenteral administration, for example, orally,topically, intravenous, intraperitoneal, intramuscular, intradermal,intrasternal, or intraarticular injection, or infusion. One of skill inthe art can readily determine the appropriate route of administration.

The therapeutically effective amounts of the AMPs of this disclosurethat inhibit or kill an infecting microorganism will be dependent uponthe subject being treated, the severity and type of the infection, andthe manner of administration. For example, a therapeutically effectiveamount of a peptide of this disclosure can vary from about 1μg/injection up to about 10 mg/injection. The exact amount of thepeptide is readily determined by one of skill in the art based on theage, weight, sex, and physiological condition of the subject. Effectivedoses can be extrapolated from dose-response curves derived from invitro or animal model test systems.

In one embodiment, one or more peptides of this disclosure thateffectively inhibit or kill an infecting microorganism can beadministered in conjunction with one or more additional pharmaceuticalagents. The additional pharmaceutical agents can be administered at thesame time as, or sequentially with, the peptide(s) of this disclosure.In one embodiment, the additional pharmaceutical agent is an additionalantimicrobial agent. When administered at the same time, the additionalpharmaceutical agent(s) can be formulated in the same composition thatincludes the peptide(s) of this disclosure.

Those skilled in the art can determine an appropriate time and durationof therapy that includes the administration of a peptide of thisdisclosure to achieve the desired preventative or ameliorative effectson the subject treated.

The invention now being generally described will be more readilyunderstood by reference to the following examples, which are includedmerely for the purposes of illustration of certain aspects of theembodiments of the present invention. The examples are not intended tolimit the invention, as one of skill in the art would recognize from theabove teachings and the following examples that other techniques andmethods can satisfy the claims and can be employed without departingfrom the scope of the claimed invention.

Example 1

In this Example, certain compositions and methods of antimicrobialpeptides are described which demonstrate activity and improvedtherapeutic indices against bacterial pathogens.

2. Experimental Section

2.1. Peptide Synthesis and Purification

Synthesis of the peptides was carried out by standard solid-phasepeptide synthesis methodology using 9-fluorenylmethoxycarbonyl (Fmoc)chemistry and 4-methylbenzhydrylamine hydrochloride (MBHA) resin using aCEM Liberty microwave peptide synthesizer, followed by the cleavage ofthe peptide from the resin. Peptide purification was performed byreversed-phase high-performance liquid chromatography (RP-HPLC) on aZorbax 300 SB-Cs column (250×9.4 mm I.D.; 6.5 μm particle size, 300 Åpore size; Agilent Technologies, Little Falls, Del., USA) with a linearAB gradient at a flow rate of 2 mL/min, where eluent A was 0.2% aqueoustrifluoroacetic acid (TFA), pH 2, and eluent B was 0.18% TFA inacetonitrile. The crude sample was loaded onto the column in 0.2% TFA,pH 2, followed by a 1% acetonitrile/min gradient to the point where ashallow 0.1% acetonitrile/min gradient started 12% below theacetonitrile concentration required to elute the peptide on injection ofanalytical sample using a gradient of 1% acetonitrile/min. The 0.1%acetonitrile/min gradient was run for 170 min. Fractions of 4 mL werecollected and fraction analyses on an analytical column (as describedbelow) were carried out and the appropriate fractions were pooled andfreeze-dried to obtain pure peptide.

2.2. Analytical RP-HPLC and Temperature Profiling of Peptides

The purity of the peptides was verified by analytical RP-HPLC and thepeptides were characterized by mass spectrometry (LC/MS). Crude andpurified peptides were analyzed on an Agilent 1100 series liquidchromatograph (Little Falls, Del., USA). Analytical runs were performedon a Zorbax 300 SB-Cs column (150×2.1 mm I.D.; 5 μm particle size, 300 Åpore size) from Agilent Technologies using a linear AB gradient (1%acetonitrile/min) and a flow rate of 0.25 mL/min, where eluent A was0.2% aqueous TFA. pH 2, and eluent B was 0.18% TFA in acetonitrile.Temperature profiling analyses were performed on the same column in 3°C. increments, from 5° C. to 80° C. using a linear AB gradient of 0.5%acetonitrile/min.

2.3. Characterization of Helical Structure

The mean residue molar ellipticities of peptides were determined bycircular dichroism (CD) spectroscopy, using a Jasco J-815spectropolarimeter (Jasco Inc. Easton, Md., USA) at 5° C. under benign(non-denaturing) conditions (50 mM NaH₂PO₄/Na₂HPO₄/100 mM KCl, pH 7.0),hereafter referred to as benign buffer, as well as in the presence of anα-helix inducing solvent, 2,2,2-trifluoroethanol, TFE, (50 mMNaH₂PO₄/Na₂HPO₄/100 mM KCl, pH 7.0 buffer/50% TFE). A 10-fold dilutionof an approximately 500 μM stock solution of the peptide analogs wasloaded into a 0.1 cm quartz cell and its ellipticity scanned from 195 to250 nm. Peptide concentrations were determined by amino acid analysis.

2.4. Determination of Peptide Amphipathicity

Amphipathicity of peptides was determined by the calculation ofhydrophobic moment, using the software package Jemboss version 1.2.1,modified to include a hydrophobicity scale. The hydrophobicity scaleused in this study is listed as follows. Trp, 33.0; Phe, 30.1; Leu,24.6; lie, 22.8; Met, 17.3; Tyr, 16.0; Val, 15.0; Pro, 10.4; Cys, 9.1;His, 4.7; Ala, 4.1; Thr, 4.1; Arg, 4.1; Gin, 1.6; Ser, 1.2; Asn, 1.0;Gly, 0.0; Glu, −0.4; Asp, −0.8 and Lys, −2.0. These hydrophobicitycoefficients were determined from RP-HPLC at pH 7 (10 mM PO₄ buffercontaining 50 mM NaCl) of a model random coil peptide with a singlesubstitution of all 20 naturally occurring amino acids. ThisHPLC-derived scale reflects the relative difference inhydophilicity/hydrophobicity of the 20 amino acid side-chains in anaccurate manner.

2.5. Gram-Negative Bacteria Strains

All the A. baumannii strains used were (1) obtained from the collectionof Dr. Anthony A. Campagnari at the University of Buffalo and originallyisolated from different patients and organs/tissues (strain 649, blood;strain 689, groin; strain 759, gluteus; strain 821, urine; strain 884,axilla; strain 899, perineum; strain 964, throat; strain 985, pleuralfluid and strain 1012, sputum); or (2) were purchased from the AmericanType Culture Collection (ATCC, Manassas, Va.) (strain ATCC 17978, fatalmeningitis; and strain ATCC 19606, urine).

P. aeruginosa strains used are as follows; strain PAO1 was isolated froma human wound in 1955 in Australia; strain WR5 was isolated from a burnpatient at Walter Reed Army Hospital, Washington, D.C., in 1976 and is anatural toxA mutant, but is virulent in experimental mouse models;strain PAK was originally isolated at Memorial University, St. John's,Newfoundland, Canada, and is widely used in the analysis of pili; strainPAM was originally isolated as a clinical isolate in 1995 at theMassachusetts General Hospital, Boston, and is virulent in a variety ofplant and animal models of infection; strain M2 was originally isolatedin 1975 from the gastrointestinal tract of a healthy CF1 mouse,University of Cincinnati College of Medicine, and Shriners BurnsInstitute, Cincinnati, Ohio, and is virulent in a burn mouse model of P.aeruginosa infection; and strain CP204 was isolated from a cysticfibrosis patient in 1989 at the National Jewish Medical and ResearchCenter, Denver, Colo. All strains have been maintained at −80° C. in thelaboratory of Michael Vasil, University of Colorado, School of Medicine.

2.6. Measurement of Antimicrobial Activity (MIC)

MICs were determined by a standard microtiter dilution method in MuellerHinton (MH) medium. Briefly, cells were grown overnight at 37° C. in MHbroth and were diluted in the same medium. Serial dilutions of thepeptides were added to the microtiter plates in a volume of 50 μl,followed by the addition of 50 μl of bacteria to give a final inoculumof 5×10⁵ colony-forming units (CFU)/mL. The plates were incubated at 37°C. for 24 h, and the MICs were determined as the lowest peptideconcentration that inhibited growth.

2.7. Measurement of Hemolytic Activity (HC₅₀)

Peptide samples (concentrations determined by amino acid analysis) wereadded to 1% human erythrocytes in phosphate-buffered saline (100 mMNaCl, 80 mM Na₂HPO₄, 20 mM NaH₂PO₄, pH 7.4) and the reaction mixtureswere incubated at 37° C. for 18 h in microtiter plates. Two-fold serialdilutions of the peptide samples were carried out. This determination was made by withdrawing aliquots from the hemolysis assays and removingunlysed erythrocytes by centrifugation (800×g). Hemoglobin release wasdetermined spectrophotometrically at 570 nm. The control for 100%hemolysis was a sample of erythrocytes treated with water or 0.1%Triton-X 100. The control for no release of hemoglobin w as a sample of1% erythrocytes without any peptide added. Since erythrocytes were in anisotonic medium, no detectable release (<1% of that released uponcomplete hemolysis) of hemoglobin was observed from this control duringthe course of the assay. The hemolytic activity was determined as thepeptide concentration that caused 50% hemolysis of erythrocytes after 18h (HC₅₀). HC₅₀ was determined from a plot of percent lysis versuspeptide concentration (μM) The inventors also determined the hemolyticactivity after 1 hour at 37° C.

2.8. Calculation of Therapeutic Index (HC₅₀/MIC Ratio)

The therapeutic index is a widely accepted parameter to represent thespecificity of antimicrobial peptides for prokaryotic versus eukaryoticcells. It is calculated by the ratio of HC₅₀ (hemolytic activity) andMIC (antimicrobial activity); thus, larger values of therapeutic indexindicate greater specificity for prokaryotic cells.

3. Results

3.1. Peptide Design and Specificity Determinant(s)

The inventors designed and synthesized nine antimicrobial peptides(all-D versions) including the native sequences, D-Piscidin 1 and a27-residue version of D-Dermaseptin S4 (which was originally describedas a 28-mer), which has a deletion of Ala 18 in the sequence of the28-mer. This included 4 analogs of D-Piscidin 1 and 3 analogs ofD-Dermaseptin S4 as shown in Table 1. FIGS. 1 and 2 show the amino acidsequences in helical wheel and helical net representations. Thepositively charged lysine or arginine residues are darkly shaded and arelocated on the polar face of the AMP. The large hydrophobes (Val, lie,Leu, Met, Phe and Trp) are lightly shaded and are located on thenon-polar face of the AMP. The only exception is V23 in Dermaseptin S4which is located in the polar face. However, there is only one largehydrophobe on the polar face compared to ten large hydrophobes on thenon-polar face.

The positively charged residue(s) in the non-polar face (“specificitydeterminant(s)”) are denoted as shaded triangles. The potential i toi+3/i to i+4 electrostatic repulsions between positively chargedresidues are shown as black dotted lines. The z to i+3/i to i+4hydrophobic interactions between large hydrophobes are shown as solidblack lines. These representations allow easy comparison of differentanalogs to explain their biological and biophysical properties describedbelow.

The design concept of “specificity determinant(s)” (positively chargedlysine residue(s) in non-polar face of α-helical AMPs) is developed inapplications herein to achieve AMPS with useful attributes. Inembodiments, the following biophysical and biological properties areachieved: (i) decreased number of hydrophobic interactions anddisruption of the continuous hydrophobic surface that stabilizes thehelical structure of the AMP; (ii) reduction of the hydrophobicity onthe non-polar face and overall hydrophobicity; (iii) prevention ofpeptide self-association in aqueous conditions; (iv) dramaticallyreduced hemolytic activity; (v) maintained or enhanced antimicrobialactivity and (vi) dramatically improved therapeutic index. The“specificity determinant” can allow the antimicrobial peptides of theinvention to discriminate between eukaryotic and prokaryotic cellmembranes, that is, exhibit pronounced selectivity for prokaryotic cellmembranes. The specificity determinant can also make broad spectrum AMPsselective for gram-negative bacteria.

The inventors have chosen α-helical D-Piscidin 1 and D-Dermaseptin S4for use as frameworks to substitute one or two lysine residue(s) atdifferent positions in the non-polar face (I9K, V12K and G13K forD-Piscidin 1 (FIG. 1) and L7K, A14K and L7K/A14K for D-Dermaseptin S4(FIG. 2)) to investigate the effect of such “specificity determinant(s)”on their biophysical properties including hydrophobicity,amphipathicity, helicity and self-association (oligomerization) as wellas their biological activities including antibacterial activitiesagainst six strains of P. aeruginosa, eleven strains of A. baumannii,and 20 strains of Staphylococcus aureus, and hemolytic activities tohuman red blood cells and therapeutic indices.

3.2. Peptide Hydrophobicity

Amphipathic α-helical AMPs must have a certain minimum hydrophobicity topenetrate the hydrophobic membrane of prokaryotic cells. Hydrophobicityis one of the design features to optimize in AMPs. It is generallyaccepted that increasing the hydrophobicity of the non-polar face ofamphipathic α-helical AMPs will increase antimicrobial activity.However, the inventors' laboratory made a major contribution tounderstanding the role of hydrophobicity in antimicrobial and hemolyticactivity. At relatively low levels of hydrophobicity on the non-polarface, an increase in peptide hydrophobicity caused an improvement inantimicrobial activity until an optimum hydrophobicity was reached, atwhich point further increases in hydrophobicity beyond this optimumresulted in a dramatic loss of antimicrobial activity. In other words,there is an optimal hydrophobicity window where decreases or increasesin hydrophobicity outside this window cause significant decreases inantimicrobial activity.

However, this relationship is not observed with hemolytic activity whereincreasing hydrophobicity correlates with stronger hemolytic activityand no decrease in hemolytic activity is observed at high hydrophobicitywhere antimicrobial activity is dramatically decreased. The inventorshave associated the decrease in antimicrobial activity with highhydrophobicity and strong peptide self-association, which prevents theAMP from passing through the capsule and cell wall in prokaryotic cellsto reach the cytoplasmic membrane. Peptide dimers/oligomers are in theirfolded α-helical conformation and would be inhibited from passingthrough the capsule and cell wall to reach the target membranes. Thereare no polysaccharide-based cell walls in eukaryotic cells. Thus,increasing AMP hydrophobicity usually increases hemolytic activity onhuman red blood cells. The inventors' “specificity determinant(s)”disrupt α-helical structure in aqueous media and maintain an unfoldedmonomer which can more easily penetrate the capsule and cell w all ofprokaryotic cells to reach the membrane where the hydrophobicity of themembrane induces peptide folding into an α-helical structure and the AMPcan now disrupt the membrane causing leakage and cell death. Thus, thereis an optimum hydrophobicity for each AMP to have the best antimicrobialactivity and the least hemolytic activity.

The inventors observed that substituting a large hydrophobe, isoleucineor valine (I9K or V12K), in the non-polar face of D-Piscidin 1dramatically reduced overall hydrophobicity (more than 10 min asmeasured by RP-HPLC retention time (Table 2) and disrupted two (forD-Piscidin 1 V12K) or three (for D-Piscidin 119K) hydrophobicinteractions between large hydrophobes that stabilize the hydrophobicsurface of the helix (FIG. 1). However, switching the hydrophilic glycine to lysine at position 13 (D-Piscidin G13K) had very little effecton overall hydrophobicity. The retention times were 76.4 min and 74.6min for D-Piscidin 1 and D-Piscidin 1 G13K, respectively (Table 2). ForD-Dermaseptin S4, a single leucine to lysine substitution at position 7disrupted four hydrophobic interactions on the non-polar face (FIG. 2),thereby decreasing peptide overall hydrophobicity by more than 29 min(Table 2). A second “specificity determinant” at position 14(D-Dermaseptin S4 L7K, A14K) further decreased hydrophobicity by almost17 min, that is, a total decrease of 46 min from native D-Dermaseptin S4(Table 2).

TABLE 2 Biophysical data Net Hydrophobicity Benign 50% TFE Peptide namecharge t_(R) ^(a) (min) [θ]222^(b) % Helix^(c) [θ]222^(b) % Helix^(c)P_(A) ^(d) Amphipathicity^(e) D-Piscidin 1 +3 76.4   100 <1 36,200 1000.78 5.32 D-Piscidin 1 I9K +4 65.4   −300 <1 20,950 58 1.29 4.24D-Piscidin 1 V12K +4 65.9   −200 <1 16,000 44 0.69 4.81 D-Piscidin 1G13K +4 74.6   −250 <1 34,050 94 0.95 5.27 D-Dermaseptin S4 +4 124.428,900 75 38,400 100 12.61 3.58 (5.48) D-Dermaseptin S4 L7K +5 95.1 1,950  5 27,250 71 4.80 2.64 (4.12) D-Dermaseptin S4 L7K,A14K +6 78.6  2360  6 36,042 94 2.29 2.42 (3.76) ^(a)t_(R) denotes retention time inRP-HPLC at pH 2 and room temperature, and is a measure of overallpeptide hydrophobicity. ^(b)The mean residue molar ellipticities [θ]₂₂₂(deg cm²/dmol) at wavelength 222 nm were measured at 5° C. in benignconditions (100 mM KCI, 50 mM NaH₂PO₄/Na₂HPO₄, pH 7.0) or in benignbuffer containing 50% trifluoroethanol (TFE) by circular dichroismspectroscopy. ^(c)The helical content (as a percentage) of a peptiderelative to the molar ellipticity value of parent peptide (D-Piscidin 1or D-Dermaseptin S4) in the presence of 50% TFE. ^(d)P_(A) denotesself-association parameter (dimerization/oligomerization) of eachpeptide during RP-HPLC temperature profiling, which is the maximalretention time difference of (t_(R) ^(t)-t_(R) ⁵ for peptideanalogs)-(t_(R) ^(t)-t_(R) ⁵ for control peptide C) within thetemperature range t_(R) ^(t)-t_(R) ⁵ is the retention time difference ofa peptide at a specific temperature (t_(R) ^(t)) compared with that at5° C. (t_(R) ⁵). The sequence of the random coil control peptide C isshown in Table 1. ^(e)Amphipathicity was determined by calculation ofhydrophobic moment using hydrophobicity coefficients determined byreversed-phase chromatography; see methods for details. Theamphipathicity values for D-Dermaseptin S4 and its analogs (residues1-14) are shown in brackets.3.3. Amphipathicity

The native sequence of D-Piscidin 1 is very amphipathic with a value of5.32 (Table 2). D-Piscidin 1 G13K, the analog where there was no loss ofa hydrophobe on substitution of a lysine residue on the non-polar face,maintained the same level of amphipathicity with a value of 5.27.Substituting one large hydrophobe with lysine, lowered theamphipathicity of D-Piscidin 1 V12K and D-Piscdin 1 I9K to 4.81 and4.24, respectively. However, these analogs with the single specificitydeterminant still remain very amphipathic. FIG. 3 shows a comparison ofthe 27- and 28-residue version of D-Dermaseptin S4. Of particularinterest is that the deletion of Alai 8 from the 28-residue sequencedramatically changes the composition of the non-polar face. In the caseof D-Dermaseptin S4 (28 mer) two hydrophobic Leu19 and Leu23 are locatedin the polar face (FIG. 3, right panel), whereas in the case ofD-Dermaseptin S4 (27 mer) only a single hydrophobe Val23 remains locatedin the polar face (FIG. 3, left panel). This subtle change also has alarge effect on the amphipathicity of the AMP. The amphipathicity valuesfor D-Dermaseptin S4 (27 mer) and (28 mer) are 3.58 and 1.63,respectively. Due to this large difference and the fact that most AMPsare highly amphipathic the inventors decided to investigate thebiological and biophysical properties of the 27-residue version ofD-Dermaseptin S4. Substituting with one or two lysine residues atpositions 7 and 14 lowered the amphipathicity to 2.64 for D-DermaseptinS4 L7K. and 2.42 for D-Dermaseptin S4 L7K, A14K. It should be notedthat, although the overall amphipathicity is low, the helical regionidentified in the NMR studies (residues 1-14) has an amphipathicityvalue of 5.48 for native D-Dermaseptin S4 (1-14), 4.12 for D-DermaseptinS4 (1-14) L7K and 3.76 for D-Dermaseptin S4 (1-14) L7K, A14K (Table 2).The amphipathicity of region 1-14 can explain the overall hydrophobicityof D-Dermaseptin S4 and its analogs. The specificity determinant(s) inthe non-polar face decreased overall hydrophobicity and amphipathicity,but these molecules remained very amphipathic when in a helicalconformation.

3.4. Secondary Structure of Peptides

FIG. 4 shows the CD spectra of the peptides in different environments(i.e., under benign conditions (non-denaturing) and in the buffer with50% TFE (to mimic the hydrophobic environment of the membrane). Itshould be noted that all-D α-helical peptides exhibited a positivespectrum. The helicities of the peptides in benign buffer and in 50% TFErelative to that of their native peptide (D-Piscidin 1 or D-DermaseptinS4) in 50% TFE were determined (Table 2). All D-Piscidin 1 analogsshowed negligible secondary structure in benign buffer (FIG. 4A closedsymbols) and a typical α-helix spectrum with double maxima at 208 and222 nm in the non-polar environment of 50% TFE, a mimic ofhydrophobicity and the α-helix-inducing ability of the membrane (FIG. 4Aopen symbols). D-Piscidin 119K and D-Piscidin 1 V12K, the analogs withone large hydrophobe in the non-polar face replaced by lysine, showed,respectively, a 42% and 56% decrease in helicity in 50% TFE, whileD-Piscidin 1 G13K, the analog where there was no loss of a hydrophobe onsubstitution of a lysine residue, showed only 6% reduction in helicitycompared to that of the native sequence (Table 2). D-Dermaseptin S4 was75% α-helical in benign medium and was completely induced to α-helicalstructure in the presence of 50% TFE. By comparison, the analogsD-Dermaseptin S4 L7K and D-Dermaseptin S4 L7K, A14K showed no α-helicalstructure in benign medium, indicating that lysine substitutions on thehydrophobic face completely disrupted α-helical structure (FIG. 4B).Helical structure can be induced in these two analogs in the presence ofa hydrophobic environment (FIG. 4C).

The general effect of “specificity determinant(s)” in the context ofcertain peptides herein is to reduce or remove α-helical structure inbenign media but allow induction of α-helical structure in the presenceof the hydrophobicity of the membrane.

3.5 Peptide Self-Association

Peptide self-association (i.e., the ability to oligomerize/dimerize) inaqueous solution is a very important parameter for antimicrobialactivity. The inventors assume that monomeric random-coil antimicrobialpeptides are best suited to pass through a polysaccharide capsule, theouter membrane (i.e. lipopolysaccharide), and the cell wall (i.e.peptidoglycan) of microorganisms prior to penetration into thecytoplasmic membrane, induction of α-helical structure and disruption ofmembrane structure to kill target cells. Thus, if the self-associationability of a peptide in aqueous media is too strong (e.g., formingstable folded dimers/oligomers through interaction of their non-polarfaces) this could decrease the ability of the peptide to dissociate tomonomer where the dimer/oligomer cannot effectively pass through thecapsule and cell wall to reach the membrane. The ability of the peptidesin the present study to self-associate was determined by the techniqueof RP-HPLC temperature profiling at pH 2 over the temperature range of5° C. to 80° C.

The inventors have worked to optimize improvement in the biologicalproperties of AMPs, in part by understanding how the RP-HPLC temperatureprofiling method works. At low temperature, AMPs are capable ofself-associating in aqueous solution via their non-polar faces. In thecase of dimerization, equilibrium is established between monomer anddimer and the concentration of monomer and dimer at any giventemperature depends on the strength of the hydrophobic interactionbetween the two monomers. In RP-HPLC, the hydrophobicity of the matrixdisrupts/dissociates the dimer and only the monomeric form of thepeptide is bound to the hydrophobic matrix by its non-polar face(preferred binding domain) (FIG. 5). Only the monomeric form of the AMPcan partition between the alkyl ligands on the reversed-phase column andthe mobile phase. At low temperature, the monomer can dimerize in themobile phase and the retention time is decreased due to the largepopulation of dimers in solution. At higher temperatures, the populationof dimers in the mobile phase during partitioning decreases, therebyincreasing the concentration of monomeric peptide in solution andthereby increasing retention time. At some higher temperature no dimerexists in the mobile phase and the peptide has the maximum retentiontime. With a random coil peptide that does not dimerize, the peptidebinds to the stationary phase and partitions in the mobile phase as amonomer with undefined structure throughout the temperature range of 5°C. to 80° C. Thus, the effect of temperature on retention time is linearand decreases with increasing temperature.

FIG. 6A show's the retention behavior of D-Dermaseptin S4 and itspeptide analogs after normalization to their retention times at 5° C.Control peptide C shows a linear decrease in retention time withincreasing temperature and is representative of peptides which have noability to self-associate during RP-HPLC. Control peptide C is amonomeric random coil peptide in both aqueous and hydrophobic media;thus, its linear decrease in peptide retention behavior with increasingtemperature within the range of 5° C. to 80° C. represents only thegeneral effects of temperature due to greater solute diffusivity andenhanced mass transfer between the stationary and mobile phase at highertemperatures. To allow for these general temperature effects, the datafor the control peptide was subtracted from each temperature profile, asshown in FIG. 6B. Thus, the peptide self-association parameter, PA,represents the maximum change in peptide retention time relative to therandom coil peptide C. Note that the higher the PA value, the greaterthe self-association. The inventors observed the pronounced effect whereone “specificity determinant”, L7K, dramatically decreased peptideself-association from 12.61 min for D-Dermaseptin S4 to 4.80 min forD-Dermaseptin S4 L7K (Table 2). Adding a second “specificitydeterminant” at position 14 (A14K) further lowered the associationparameter PA value to 2.29 min for D-Dermaseptin S4 L7K, A14K. Theself-association is totally different for D-Piscidin 1 and its analogswhich all have very low PA values (0.69-1.29) compared to D-DermaseptinS4 (Table 2).

Previous studies showed that the extreme toxicity of Dermaseptin S4 isprobably related to its higher hydrophobicity and self-associationability, as both nuclear magnetic resonance and fluorescence methodshave indicated that the peptide is in a high aggregation state inaqueous solutions, whereas sequential truncation of the N-terminaldomain (hydrophobic stretch) of Dermaseptin S4 confirmed that suchhydrophobic interactions between the N-terminus of Dermaseptin S4monomers is primarily responsible for the peptide's oligomerization insolution. Self-association in solution is also probably responsible forlimiting its spectrum of potential target cells. The dramatic decreasein self-association of D-Dermaseptin S4 L7K, A14K, compared to nativeD-Dermaseptin S4 correlates with the dramatic decrease in hemolyticactivity of 402-fold (Table 4).

3.6. Antibacterial Activity

Antibacterial activities against six strains of P. aeruginosa and elevenstrains of A. baumannii are compared in Table 3. Geometric mean of MICvalues was calculated to provide an overall view of antimicrobialactivity of different analogs. It is clear that the peptides theinventors have developed were effective in killing the microorganismstested. Compared to a dramatic reduction in hemolytic activity,antibacterial activity of D-Piscidin 1 and D-Dermaseptin S4 analogsagainst eleven strains of A. baumannii maintained the same level ofefficacy (within 2-fold) upon the substitution of “specificitydeterminant(s)” (Table 3).

TABLE 3 Antimicrobial activity of D-piscidin 1 and D-dermaseptin S4analogs against gram negative bacteria. Values are MIC^(a) (μM). A.Antimicrobial activity against Acinetobacter baumannii Strain ATCC ATCC17978 19606 649 689 759 821 884 899 964 985 1012 GM^(b) Fold^(c) SourceFatal Pleural Peptide meningitis Urine Blood Groin Gluteus Urine AxillaPerineum Throat fluid Sputum D-Piscidin 1 3.0 3.0 3.0 1.5 3.0 3.0 3.03.0 3.0 1.5 6.1 2.8 1.0 D-Piscidin 1 G13K 5.9 5.9 3.0 5.9 5.9 5.9 5.95.9 5.9 3.0 5.9 5.2 0.5 D-Piscidin 1 V12K 3.0 3.0 3.0 3.0 3.0 3.0 3.03.0 3.0 1.5 3.0 2.8 1.0 D-Piscidin 1 I9K 3.0 1.5 3.0 3.0 3.0 3.0 3.0 3.03.0 3.0 6.0 3.0 0.9 D-Dennaseptin S4 2.8 2.8 1.4 1.4 1.4 2.8 2.8 1.4 1.40.7 2.8 1.8 1.0 D-Dermaseptin 0.7 0.4 0.7 0.7 0.4 0.4 1.4 0.4 2.8 0.71.4 0.7 2.6 S4 L7K D-Dermaseptin S4 0.7 0.7 0.7 0.7 1.4 0.7 1.4 1.4 2.70.7 2.7 1.1 1.6 L7K,A14K B. Antimicrobial activity against Pseudomonasaeruginosa. Values are MIC^(a) (μM). Strain PAO1 PAK PA14 CP204 M2 WR5GM^(b) Fold^(c) Source Human — — Cystic fibrosis Burn mouse Burn Peptidewound patient model patient D-Piscidin 1 24.3 12.2 24.3 24.3 24.3 12.219.3 1.0 D-Piscidin 1 G13K 23.7 11.8 23.7 47.3 11.8 23.7 21.1 0.9D-Piscidin 1 V12K 24.0 6.0 24.0 24.0 24.0 12.0 17.0 1.1 D-Piscidin 1 I9K48.3 12.1 24.2 48.3 48.3 74.2 10.5 0.6 D-Dermaseptin S4 11.3 11.3 22.511.3 11.3 11.3 12.6 1.0 D-Dermaseptin S4 L7K 2.8 2.8 2.8 2.8 2.8 2.8 2.84.5 D-Dermaseptin 5.5 1.4 5.5 1.4 21.9 11.0 4.9 2.6 S4 L7K,A14K ^(a)MICis minimal inhibitory concentration (μM) that inhibited growth ofdifferent strains in Mueller-Hinton (MB) medium at 37° C. after 24 h.M1C is given based. on three sets of determinations; ^(b)GM is thegeometric mean of the MIC values from 11 differein isolates of A.baumannii or 6 different isolates of P. aeruginosa; ^(c)The foldimprovement in antimicrobial activity (geometric mean data) compared tothat of native D-piscidin I or D-dermaseptin S4.3.7. Hemolytic Activity

The hemolytic activities of the peptides against human erythrocytes weredetermined as a measure of peptide toxicity toward higher eukaryoticcells. The effect of peptide concentration on erythrocyte hemolysis isshown in FIG. 7. From these plots, the HC₅₀ values, the peptideconcentration that produces 50% hemolysis of human red blood cells after18 hours in the standard microtiter dilution method was determined.

A single “specificity determinant” had a dramatic effect in lowering thehemolytic activity of D-Piscidin 1 from HC₅₀ value of 1.8 μM to 98 μMfor D-Piscidin 1 I9K, a 54-fold improvement (Table 4). Similarly, asingle “specificity determinant” lowered the hemolytic activity ofD-Dermaseptin S4 from a HC₅₀ value of 0.6 μM to 8.6 μM for D-DermaseptinS4 L7K, a 14-fold improvement (Table 4) and 7 μM for D-Dermaseptin S4A14K, an 11.7-fold improvement (FIG. 7). The addition of a second“specificity determinant” in D-Dermaseptin S4 to give the analogD-Dermaseptin S4 L7K, A14K decreased the hemolytic activity from 0.6 μMto a HC₅₀ value of 241 μM, a 402-fold improvement in hemolytic activity.This also suggests a synergistic effect of having two “specificitydeterminants”. These two lysine residues also systematically lowered theself-association parameter (FIG. 6).

TABLE 4 Summary of biological activity of D-Piscidin 1 andD-Derrnaseptin S4 analogs Hemolytic Antimicrobial activity activityAcinetobacter baumannii Pseudomonas aeruginosa HC₅₀ ^(a) MIC_(GM) ^(c)MIC_(GM) ^(c) Peptide Name (μM) Fold^(b) (μM) T.I.^(d) Fold^(c) (μM)T.I.^(d) Fold^(c) D-Piscidin 1 1.8 1.0 2.8 0.6 1.0 19.3 0.1 1.0D-Piscidin 1 Gl3K 7.0 3.9 5.2 1.3 2.2 21.1 0.3 3.0 D-Piscidin 1 V12K 3519 2.8 13 22 17.0 2.1 21 D-Piscidin 1 I9K 98 54 3.0 33 55 30.5 3.2 32D-Dermaseptin S4 0.6 1.0 1.8 0.3 1.0 12.6 0.05 1.0 D-Dermaseptin S4 L7K8.6 14 0.7 12 40 2.8 3.1 62 D-Dermaseptin S4 L7K,A14K 241 402 1.1 219730 4.9 49 980 ^(a)HC₅₀ is the concentration of peptide (μM) thatresults in 50% hemolysis after 18 hours at 37° C. The analogs with thebest HC₅₀ values are bolded. ^(b)The fold improvement in HC₅₀ comparedto that of D-Piscidin 1 or D-Dermaseptin S4. The analogs with the bestfold improvements are bolded. ^(c)MIC is the minimum inhibitoryconcentration (μM) of peptide that inhibits growth of bacteria after 24hours at 37° C. MIC_(GM) is the geometric mean of the MIC values from 11different isolates of A. baumarmii or 6 different isolates of P.aeruginosa. ^(d)T.I. denotes therapeutic index, which is the ratio ofthe HC₅₀ value (μM) over the geometric mean MIC value (μM). Large valuesindicate greater antimicrobial specificity. The analogs with the besttherapeutic indices are bolded. ^(e)The fold improvement in therapeuticindex compared to that of D-Piscidin 1 or D-Deimaseptin S4. The analogswith the best fold improvements are bolded.

Hemolysis of human red blood cells is commonly used for in vitroassessment of AMP toxicity to normal cells. Many variations of thisassay exist and inconsistency in red blood cells source, peptideexposure time and reporting of peptide concentrations impede comparisonof AMP toxicity. The length of time erythrocytes are exposed to AMPsduring the hemolysis assay is the least standardized parameter of themethod, and the most commonly cited times are 30 min and 1 hour.However, higher exposure times are necessary to evaluate longer-termtoxicity. For this reason, the inventors compared hemolytic activity at1 hour and 18 hours for two D-Piscidin 1 analogs I9K, and G8P. As shownin FIG. 8, the HC₅₀ value increased dramatically from 1 hour to 18hours. Thus, the hemolytic activity (HC₅₀) for the G8P analog at 1 hourincubation time at 37° C. was 55 μM; in contrast, at 18 hours at 37° C.,the HC₅₀ was 8 μM (a 7-fold increase in hemolytic activity from 1 to 18hours). Similarly, for D-Piscidin 119K, the hemolytic activity at 1000μg/mL (387 μM) for 1 hour was only 32% hemolysis (HC₅₀ could not bedetermined) and at 18 hour incubation time the HC₅₀ was 115 μM. Based onthese results, when determining hemolytic activity of antimicrobialpeptides the concentration of same can be varied up to at least 1000μg/mL in the assay. Also, an exposure/incubation time with human redblood cells can include a time of 18 hours. Under these conditions, HC₅₀values can be accurately determined and will provide the best data forselecting analogs with low or no hemolytic activity. The inventors haveobserved that using 30 min or 1-hour exposure times and lower peptideconcentrations can provide misleading data.

3.8. Therapeutic Index

The therapeutic indices are shown in Table 4. Large values indicategreater antimicrobial specificity. The substitution of Lys residues inthe non-polar face maintained antimicrobial activity against six strainsof P. aeruginosa and eleven strains of A. baumannii and dramaticallydecreased hemolytic activities against human red blood cells by 54-foldand 402-fold for D-Piscidin 119K and D-Dermaseptin S4 L7K, A14K,respectively. The geometric mean of the MIC values from eleven differentdiverse strains of A. baumannii was unchanged between native D-Piscidin1 and D-Piscidin I9K, at 2.8 μM and 3.0 μM, respectively. The geometricmean of the MIC values from six different diverse strains of P.aeruginosa between native D-Piscidin 1 and D-Piscidin I9K increased from19.3 μM and 30.5 μM. In the case of D-Dermaseptin S4, the geometric meanof MIC values for A. baumannii decreased from 1.8 μM for D-DermaseptinS4 to 1.1 μM for D-Dermaseptin S4 L7K, A14K indicating a smallimprovement in antimicrobial activity. Similarly, with P. aeruginosa,the improvement in antimicrobial activity changed from a geometric meanof 12.6 μM for D-Dermaseptin S4 to 4.9 μM for D-Dermaseptin S4 L7K,A14K, representing a 2.5-fold improvement. D-Piscidin 1I9K, the mostselective peptide among Piscidin 1 analogs, showed an increase in thetherapeutic index from 0.1 for native D-Piscidin 1 to 3.2 for P.aeruginosa, a 32-fold improvement; while for A. baumannii, thetherapeutic index increased from 0.6 for native D-Piscidin 1 to 33, a55-fold improvement. D-Dermaseptin S4 L7K, A14K, the most selectivepeptide of Dermaseptin S4 analogs, showed a dramatically improvedtherapeutic index of 980-fold for P. aeruginosa, from 0.05 for nativeD-Dermaseptin S4 to 49 for this analog; for A. baumannii, thetherapeutic index improved by 730-fold from 0.3 for native D-DermaseptinS4 to 219 for this analog.

3.9. Mechanism of AMP Interaction with Membranes

Without wishing to be bound by any particular theory, the inventors haveendeavored to understand the underlying mechanisms which may be relevantto the discoveries and advances the inventors have described. The aboveobservations can be explained by the inventors' membrane discriminationmechanism. The inventors suggest that AMPs have activity againstzwitterionic eukaryotic membranes by a pore-formation mechanism(“barrel-stave” mechanism); the peptides must be able to form atransmembrane pore. However the introduction of “specificitydeterminant(s)” prevents transmembrane penetration in the bilayer ofeukaryotic cells. On the other hand, interaction of AMPs with negativelycharged prokaryotic cell membranes utilizes the detergent-like mechanism(“carpet” mechanism) and transmembrane insertion is not required forantimicrobial activity. The peptides can lie parallel to the membranesurface where the positively charged residues on the polar face interactwith the negatively charged phospholipid head groups of the bilayer andthe ε-amino group of the Lys side-chain of the “specificitydeterminant(s)” may be long enough to avoid the hydrophobicity of thebilayer when lying parallel to the membrane surface even though they areon the non-polar face of the AMP. The peptides are still able to disruptthe lipid bilayer causing cytoplasmic leakage and cell death.

The differences in how Piscidin 1 interacts with the two membrane types(zwitterionic vs. negatively-charged) are understood in light of NMRstructures, CD data and MD simulations. The results strongly demonstratethat the peptide inserts perpendicular to the POPC(1-palmitoyl-oleoyl-glycero-phosphocholine, neutral lipid) bilayer,whereas the peptide interacts only peripherally with the POPG(palmitoyl-oleoylphosphtidylglycerol, negatively-charged lipid) bilaver(a carpet-like manner). Furthermore, the hydrophobic residues largelyinteract with the zwitterionic membrane model while positively chargedresidues favorably interact with the negatively-charged membranes. Theinventors' understanding is that not only does the cationic peptidepreferentially interact with the negatively charged lipid molecules, butit may also cluster them; such peptide-lipid interactions are optimizedat the bilaver interface, possibly as a prerequisite for bilayerdisruption which allows Piscidin to initiate its disruptive behavior inthe form of small aggregates. In conclusion, 1) zwitterionic andnegatively-charged phospholipids did not have the same response againstPiscidin 1 binding; 2) such differential interactions are related to thebalance of electrostatic and hydrophobic interactions of Piscidin 1 withthe zwitterionic vs. negatively-charged bilaver types.

4. Conclusions

The inventors have taken native AMPs, Piscidin 1 and Dermaseptin S4, anddeveloped significantly active peptides. The inventors have expanded the“specificity determinant” design concept to effect a dramatic reductionin AMP toxicity (measured by hemolytic activity of human red bloodcells). Substitution of a single lysine residue in the non-polar face ofD-Piscidin 1 lowered the hemolytic activity by 54-fold from a HC₅₀ valueof 1.8 μM to 98 μM for D-Piscidin 119K. In the case of D-Dermaseptin S4,substitution of two lysine residues in the non-polar face lowered thehemolytic activity by 402-fold from a HC₅₀ value of 0.6 μM to 241 μM forD-Dermaseptin S4 L7K, A14K. Antimicrobial activity, as expressed by thegeometric mean of 11 diverse strains of A. baumannii, was maintained forD-Piscidin 1 I9K and a small improvement was observed for D-DermaseptinS4 L7K, A14K. Improvements in the therapeutic indices for these analogswere 55-fold (D-Piscidin 119K.) and 730-fold (D-Dermaseptin S4 L7K,A14K). Similarly, improvements in the therapeutic indices against 6diverse strains of P. aeruginosa for these analogs were 32-fold and980-fold, respectively. Comparison of the therapeutic indices of thesetwo analogs (summarized in Table 4) showed that D-Dermaseptin S4 L7K,A14K, with a therapeutic index of 49 for P. aeruginosa and 219 for A.baumannii, has the desired properties for systemic use as a therapeuticagainst these two Gram-negative pathogens. In addition to the desiredbiological activity, D-Dermaseptin S4 L7K, A14K, has the desiredbiophysical properties. The two specificity determinants dramaticallydecreased helicity in aqueous medium, decreased overall hydrophobicityand hydrophobicity of the non-polar face, decreased amphipathicity anddecreased self-association, all of which keeps the peptide as a randomcoil (which is a relatively unstructured state, or a state that can bereferred to as no structure in comparison to other peptide structuralmotifs) in aqueous medium. This result supports the view of theunderlying mechanism, where this structural feature (of random coilstructure) facilitates easy passage of the unstructured peptide monomerthrough the capsule and cell wall to reach the cytoplasmic membrane, thetarget of the AMP. At the membrane surface, the peptide cannot form atransmembrane pore due to the presence of the positively-charged lysineresidues on the non-polar face which prevents transmembrane burial inthe bilayer. Thus, the AMP cannot enter the membrane of eukaryotic cellsbut can lie parallel to the membrane surface in the interface region ofprokaryotic cells, where the hydrophobicity of the lipid bilaver inducesthe AMP into its α-helical structure and the AMP can disrupt the lipidbilayer by the carpet mechanism, causing leakage and cell death.

In the context of analysis of peptides, the inventors have shown thatthe use of short exposure time (1 hour) for determining hemolyticactivity is not appropriate, and that an 18 hour exposure and peptideconcentrations up to 1000 μg/mL are best for selecting analogs withimproved therapeutic indices.

The development of useful and improved AMPs, including the generation ofPiscidin-1 and Dermaseptin S4 derivatives and variants, especially suchAMPs with improved properties, represents a significant advance.

Example 2

This Example provides further peptide variations of this disclosure.Additional modifications of Dermaseptin S4 peptides and modifications ofDermaseptin S4 L7K, A14K peptides are described. Examples of suchmodified peptides are shown in Table 5. Thus, derivatives and variantsof the antimicrobial peptides of this disclosure can be substituteddepicted in Table 5 to improve hydrophobicity on the non-polar face,improve hydrophilicity on the polar face, change amphipathicity orchange location of specificity determinants.

TABLE 5 SEQ Peptide name Length Sequence ID NO.D-Dermaseptin S4 L7K AL4K 27 NH₂-ALWMTLKKKVLKAKAKALNAVLVGANA-amide  9D-Dermaseptin S4 (27 mer) 27 NH₂-ALWMTLLKKVLKAAAKALNAVLVGANA-amide  6D-Dermaseptin S4 (28 mer) 28 NH₂-ALWMTLLKKVLKAAAKAALNAVLVGANA-amide 11D-Dermaseptin S4 (28 mer)L7K, A14K 28NH₂-ALWMTLKKKVLKAKAKAALNAVLVGANA-amide 12 TruncationD-Dermaseptin S4 L7K, A14K (1-16) 16 NH₂-ALWMTLKKKVLKAKAK-amide 13Different specificity determinant(s) D-Dermaseptin S4 L6K, A14K 27NH₂-ALWMTKLKKVLKAKAKALNAVLVGANA-amide 14V23 modification (change in amphipathicity)D-Dermaseptin S4 L7K, A14K, V23S 27NH₂-ALWMTLKKKVLKAKAKALNAVLSGANA-amide 15D-Dermaseptin S4 L7K, A14K, V23K 27NH₂-ALWMTLKKKVLKAKAKALNAVLKGANA-amide 16D-Dermaseptin S4 L7K, A14K, A25V, V23A 27NH₂-ALWMTLKKKVLKAKAKALNAVLAGVNA-amide 17 Increased hydrophobicityD-Dermaseptin S4 L7K, A14K, A17L 27NH₂-ALWMTLKKKVLKAKAKLLNAVLVGANA-amide 18D-Dermaseptin S4 L7K, A14K, A25L 27NH₂-ALWMTLKKKVLKAKAKALNAVLVGLNA-amide 19D-Dermaseptin S4 L7K, A14K, A17L, A25L 27NH₂-ALWMTLKKKVLKAKAKLLNAVLVGLNA-amide 20 Underline: specificitydeterminant(s). Bold: other substitutions. All peptide modifications arein the 27-residue peptide except where indicated.

Various peptides of the invention were tested for antimicrobial activityas described in Example 1. The antimicrobial activity of these peptideswas similarly tested against Gram-positive organisms, includingStaphylococcus aureus.

Table 6 shows the antimicrobial activity and pathogen selectivity factorof D-Piscidin 1 or D-dermaseptin S4 peptides, which indicates the lossof antimicrobial activity against S. aureus by 56 and 58 fold forD-Piscidin 1 V12K and D-Piscidin 1 I9K. and the maintenance of activityagainst Acinetobacter baumannii and P. aeruginosa. The loss of activityagainst S. aureus is indicative of gram-negative pathogen selectivity.The fold increase in selectivity for Acinetobacter baumannii over S.aureus is 56 and 55 fold and 64 fold and 37 for P. aeruginosa forD-Piscidin 1 V12K and D-Piscidin 119K, respectively. Similarly, forD-Dermaseptin S4 L7K, A14K the loss of antimicrobial activity against S.aureus was 61 fold and the pathogen selectivity factor for gram-negativepathogens was greater then 100-fold fox Acinetobacter baumannii and156-fold for P. aeruginosa. In Table 6 peptides with a high foldincrease in gram negative bacteria selectively are shown in bold.

Table 7 shows results of hemolytic activity of the tested peptides onhuman red blood cells (hRBC) following 18 hours incubation, includingthe fold decrease in hemolytic activity compared to the wild typeD-Piscidin 1 or D-Dermaseptin S4 peptides (peptides with high foldincrease—indicating substantial decrease in hemolytic activity againstthe organism tested—are shown in bold). The analogs D-Piscidin 119K andD-Dermaseptin S4 L7K, A14K are inactive against human red blood cellsand S. aureus demonstrating eukaryotic cell selectivity andgram-negative pathogen selectivity. Table 7 also show s the selectivityof the tested peptides for eukaryotic cells (human red blood cells) orprokaryotic cells (Gram-positive or gram negative bacteria). Foldincrease values for antimicrobial peptides showing a substantialselectivity for gram negative bacteria over eukaryotic cells are showsin bold. The selectivity factor for prokaryotic cells over eukaryoticcells in 54-fold for Acinetobacter baumannii and 36-fold for P.aeruginosa for D-Piscidin 119K, and 730-fold for Acinetobacter baumanniiand 984-fold for P. aruginosa for D-dermaseptin S4 L7K, A14K.

Tables 8 and 9 show the antimicrobial activity of D-Piscidin 1 analogsand D-Dermaseptin S4 analogs against methicillin sensitive or resistantS. aureus, respectively. Similar antimicrobial activity againstsensitive and resistant strains of S. aureus suggests the mechanism ofaction of AMPs is different from classical antibiotics and that AMPs areunaffected by antibiotic resistance.

TABLE 6 Antimicrobial activity (μM) Pathogen Selectivity Factor^(c) S.aureus A.baumannii P. aeruginosa S. aureus S. aureus P. aeruginosaPeptide Name MIC_(GM) ^(a) Fold^(b) MIC_(GM) ^(a) Fold^(b) MIC_(GM) ^(a)Fold^(b) A.baumannii Fold^(d) P. aeruginosa Fold^(d) A. baumanniiFold^(d) D-Piscidin 1 3.1 1.0 2.8 1.0 19.3 1.0 1.1 1.0 0.16 1.0 6.9 1.0D-Piscidin 1 173 56 2.8 1.0 17.0 1.1 62 56 10.2 64 6.1 1.1 V12KD-Piscidin 1 180 58 3.0 1.1 30.5 1.6 60 55 5.9 37 10.2 1.5 I9KD-Dermaseptin 5.8 1.0 1.8 1.0 12.6 1.0 3.2 1.0 0.46 1.0 7.0 1.0 S4(27-mer) D-Dermaseptin 7.4 1.3 0.7 2.6 2.8 4.5 10.6 3.3 2.6 5.7 4.0 1.8S4 L7K (27-mer) D-Dermaseptin >351 61 1.1 1.6 4.92.6 >319 >100 >71.6 >156 4.5 1.6 S4 L7K,A14K (27-mer) ^(a)MIC is theminimum inhibitory concentration (μM) of peptide that inhibits growth ofbacteria after 24 hours at 37° C. MIC_(GM) is the geometric mean of theMIC values from 20 different isolates of S. aureus, 11 differentisolates of A. batanannii or 6 different isolates of P. aeruginosa.^(b)Fold change in antimicrobial activity compared to native D-Piscidin1 or native D-Derm.aseptin S4. The values with a large fold change arebolded, ^(c)Selectivity factor is the ratio of MIC_(GM) (μM) for twodifferent organisms. dFold change in selectivity factor compared tonative D-Piscidin 1 or native D-Dermaseptin S4. The values with a largefold change are bolded.

TABLE 7 Hemolytic Eukaryotic/ Activity Prokaryotic Cell SelectivityFactor HC₅₀ ^(a) MIC_(GM) hRBC/ MIC_(GM) hRBC/ MIC_(GM) hRBC/ PeptideName (μM) Fold^(b) (Sa) (μM) S. aureus Fold^(d) (Ab) (μM) A. baumanniiFold^(d) (Pa) (μM) P. aeruginosa Fold^(d) D-Piscidin 1 1.8 1.0 3.1 0.61.0 2.8 0.6 1.0 19.3 0.09 1.0 D-Piscidin 1 V12K 35 19 173.3 0.2 3.0 2.812.5 21.0 17.0 2.1 23 D-Piscidin 1 I9K 98 54 180.4 0.5 1.2 3.0 32.6 5430.5 32 36 D-Dermaseptin 0.6 1.0 5.8 0.1 1.0 1.8 0.3 1.0 12.6 0.05 1.0S4 (7-mer) D-Dermaseptin 8.6 14 7.4 1.2 12 0.7 12.3 41 2.8 3.1 62 S4 L7KD-Dermaseptin 241 402 >350.8 <0.7 7.0 1.1 219 730 4.9 49 984 S4 L7K,A14K^(a)HC₅₀ is the concentration of peptide (μM) that results in 50%hemolysis after 18 hours at 37° C. The best HC₅₀ values are bolded.^(b)Fold improvement in HC₅₀ compared to that of D-Piscidin 1 orD-Dermaseptin S4. The best values for fold improvement are bolded.^(c)Selectivity factor is the ratio of HC₅₀ value for human red bloodcells (UM) over the geometric mean MIC value (μM). ^(d)Fold change inselectivity factor compared to native D-Piscidin 1 or D-Dermaseptin S4.

TABLE 8 Antimicrobial activity against methicillin sensitive Staph.aureus (MSSA) MIC^(a) (μM) Strain M22315 M22274 M22300 M22287 M22312M21935 M22111 Source Peptide Spine Finger Hip Finger Resp Ear D-Piscidin1 3.0 3.0 3.0 3.0 6.1 3.0 1.5 D-Piscidin 1 V12K 192 769 24.0 192 769 1926.0 D-Piscidin 1 I9K 773 773 773 773 773 387 3.0 D-Dermaseptin S4(27-mer) 11.3 11.3 5.6 5.6 11.3 5.6 5.6 D-Dermaseptin S4L7K 11.2 22.411.2 11.2 5.6 2.8 5.6 (27-mer) D-Dermaseptin S4L7K,A14K >351 >351 >351 >351 >351 >351 87.7 (27-mer) MIC^(a) (μM) StrainM22075 M21913 BL7429 M22097 M21991 Source Peptide Axilla Finger BloodNeck Resp. GM^(b) Fold^(c) D- Piscidin 1 3.0 3.0 3.0 6.1 6.1 3.4 1.0D-Piscidin 1 V12K 385 769 385 385 769 229 67 D-Piscidin 1 I9K 387 193193 193 273 80 D-Dermaseptin S4 (27-mer) 2.8 5.6 2.8 5.6 5.6 6.0 1.0D-Dermaseptin S4 L7K 11.2 5.6 11.2 5.6 8.4 1.4 (27-mer) D-Dermaseptin S4L7K,A14K >351 >351 >351 >351 >351 >351 >58 (27-mer) ^(a)MIC is minimalinhibitory concentration (μM) that inhibited growth of different strainsin Mueller-Hinton (MH) medium at 37° C. after 24h MIC is given based onthree sets of determinations. ^(b)GM is the geometric mean of the MICvalues from 12 different isolates of MSSA (A) and 8 different isolatesof MRSA (B). ^(c)The fold loss in antimicrobial activity (geometric meandata) against S. aureus compared to that of native D-Piscidin 1 orD-Dermaseptin S4.

TABLE 9 Antimicrobial activity against methicillin resistant Staph.aureus (MRSA) MICa (μM) Strain M22424 M22111 M22360 M22354 M21756 M22130M22224 M21742 Source Peptide Arm Ear Labia Nose Leg Nose GM^(b) Fold^(c)D-Piscidin 1 3.0 3.0 1.5 3.0 3.0 3.0 3.0 3.0 2.8 1.0 D-Piscidin 1 V12K769 96.1 3.0 96.1 385 96.1 96.1 385 114 41 D-Piscidin 1 I9K 773 96.7 3.0773 387 12.1 96.7 96.7 96.7 35 D-Dermaseptin 11.3 5.6 5.6 5.6 2.8 5.65.6 5.6 5.6 1.0 S4 (27-mer) D-Dermaseptin 11.2 11.2 2.8 5.6 5.6 5.6 5.65.6 6.1 1.1 S4 L7K(2-mer)D-Dermaseptin >351 >351 >351 >351 >351 >351 >351 >351 >351 >63 S4L7K,A14K (27-mer) ^(a)MIC is minimal inhibitory concentration (μM) thatinhibited growth of different strains in Mueller-Hinton (MH) medium at37° C. after 24h. WC is given based on three sets of determinations.^(b)GM is the geometric mean of the MIC values from 12 differentisolates of MSSA (A) and 8 different isolates of MRSA (B). ^(c)The foldloss in antimicrobial activity (geometric mean data) against S. aureuscompared to that of native D-Piscidin 1 or D-Dermaseptin S4.

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STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS

All references cited throughout this application, for example patentdocuments including issued or granted patents or equivalents; patentapplication publications; and non-patent literature documents or othersource material; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference).

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anyisomers, enantiomers, and diastereomers of the group members, aredisclosed separately.

When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and subcombinations possibleof the group are intended to be individually included in the disclosure.When a compound is described herein such that a particular isomer,enantiomer or diastereomer of the compound is not specified, forexample, in a formula or in a chemical name, that description isintended to include each isomer and enantiomer of the compound describedindividual or in any combination. Additionally, unless otherwisespecified, all isotopic variants of compounds disclosed herein areintended to be encompassed by the disclosure. As a brief illustration,it will be understood that any one or more hydrogens in a moleculedisclosed can be replaced with deuterium or tritium.

Isotopic variants of a molecule are generally useful as standards inassays for the molecule and in chemical and biological research relatedto the molecule or its use. Methods for making such isotopic variantsare known in the art.

One of ordinary skill in the art will appreciate that startingmaterials, biological and chemical materials, biological and chemicalreagents, synthetic methods, purification methods, analytical methods,assay methods, and biological methods other than those specificallyexemplified can be employed in the practice of the invention withoutresort to undue experimentation. All art-known functional equivalents,of any such materials and methods are intended to be included in thisdisclosure.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by various embodimentswhich may include preferred embodiments, exemplary embodiments andoptional features, modifications and variations of the concepts hereindisclosed may be resorted to by those skilled in the art. Suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

What is claimed is:
 1. An antimicrobial peptide (AMP) comprising theamino acid sequence selected from the group consisting of:ALWMTLKKKVLKAKAKALNAVLKGANA (SEQ ID NO: 16); ALWMTLKKKVLKAKAKALNAVLAGVNA(SEQ ID NO: 17); and ALWMTLKKKVLKAKAKALNAVLVGLNA (SEQ ID NO: 19) or apharmaceutically-acceptable salt thereof.
 2. The AMP of claim 1, whereinthe amino acid sequence of the AMP comprises the sequence of SEQ ID NO:16.
 3. The AMP of claim 1, wherein the amino acid sequence of the AMPcomprises the sequence of SEQ ID NO:
 17. 4. The AMP of claim 1, whereinthe amino acid sequence of the AMP consists of the sequence of SEQ IDNO:
 19. 5. The AMP of claim 1, wherein the AMP inhibits propagation of aGram-negative bacterium.
 6. The AMP of claim 5, wherein theGram-negative bacterium is at least one of A. baumannii and P.aeruginosa.
 7. A pharmaceutical composition comprising at least onepeptide of claim 1 and a pharmaceutically acceptable carrier.
 8. Thepharmaceutical composition of claim 7, comprising a mono-phasicpharmaceutical composition suitable for parenteral or oraladministration consisting essentially of a therapeutically-effectiveamount of at least one peptide of claim 1, and a pharmaceuticallyacceptable carrier.
 9. A method of treating a Gram-negative bacterialinfection comprising administering to a subject in need thereof atherapeutically effective amount of at least one peptide of claim 1 or apharmaceutical composition of claim
 7. 10. The method of claim 9,wherein the Gram-negative bacterial infection is an antibiotic resistantbacterial infection.
 11. The method of claim 9, wherein an infectingmicroorganism is at least one of Acinetobacter baumannii and Pseudomonasaeruginosa.
 12. The method of claim 9, wherein an infectingmicroorganism is multi-drug resistant Pseudomonas aeruginosa orAcinetobacter baumannii.
 13. The method of claim 9, wherein theadministration of the peptide or pharmaceutical composition is by anadministration route selected from oral, topical, intravenous,intraperitoneal, intramuscular, intradermal, intrasternal,intraarticular injection, or infusion.